JP3436756B2 - Meningococcal class I outer membrane protein vaccine - Google Patents

Abstract

Outer-membrane vesicles, Class 1 outer membrane proteins of Neisseria meningitidis, fragments or oligopeptides containing epitopes of the Class 1 I OMP can be used to immunize against meningococcal disease.

Description

BACKGROUND OF THE INVENTION Bacterial meningitis is a disease of central nervous system inflammation caused by the growth of bacteria in or adjacent to pialitis. Meningitis is an acute infection that affects children and young adults and is caused by Neisseria meningitidis, which among other factors includes other bacterial and viral pathogens.

N. meningitidis is subdivided into serological groups depending on the presence of capsular or cell wall antigens. The currently recognized serogroups are A, B, C, as separated by serum aggregation.
D, W-1, X, Y, Z, and 29E. The polysaccharides responsible for the specificity of the serogroups of groups A, B, C, W-135 and Y were purified.

The proportion of carriers of N. meningitidis is much higher than the incidence of the disease. Some are temporary carriers while others are chronic carriers, releasing meningococci more or less continuously or in some occasional fashion. The meningococcal carrier status is a process of immunization, and within 2 weeks of colonization, production of antibodies to N. meningitidis can be identified. Bacterial antibodies appear to be directed against both capsular polysaccharides and other cell wall antigens.

Studies have shown that the outer membrane of N. meningitidis has 3-5 major proteins, with the major 41,000 or 38,000 Mr proteins having serotype-specific antigenic determinants.
There is a considerable degree of strain heterogeneity in the profile of outer membrane proteins on sodium dodecyl sulfate-polyacrylamide electrophoretic gels (SDS-PAGE).
As defined by the chain of peptide mapping, proteins consist of five classes, designated 1-5, based on common peptide structure. Bacterial monoclonal antibodies were raised against the 46,000 Mr class I protein, which is partly shared between the structures of different serotypes. [Frasch, CE et al. (1985) p. 63
3, "New developments in meningococcal vaccines (New De
velopments in Meningococcal Vaccines) ", GK
Schoolnik et al. (Ed.), The Pathogenic Nisseria American Society for Microbiology (American)
Society for Microbiology), Washington, DC].

Vaccines against organisms have been developed using the capsular polysaccharides of group A, C, W-135 or Y. Although these vaccines were effective for a short period of time, they did not elicit immunological memory, and subjects had to be vaccinated again within almost 3 years to maintain their resistance . Group B polysaccharides were poorly immunogenic and no successful vaccine was produced. A possible explanation for the low activity was resistance to group B polysaccharides elicited by cross-reactive antigens found in human tissues, such as the brain. Furthermore, in the study,
The majority of bacterial antibodies in the convalescent sera of patients with group B Myelobacteriaceae disease are directed against outer membrane proteins.

A vaccine for protection against Group B meningococcal disease was developed in which a non-covalently bound complex of outer membrane protein (OMP) and a Group B polysaccharide were administered. Beuvery et al. (1983) Infect. Immun. 40 : 369-380. However, B polysaccharide elicits a transient IgM antigen response, which does not confer immunoprotection. In addition, there is large antigenic diversity and variability in the N. meningitidis outer membrane protein from strain to strain. Moreover, lipopolysaccharides are present in OMPs and exhibit good antigenic variability as well.

There is a need for a safe and effective vaccine against meningococcal disease that provides immunity from infections, especially in infants and adults.

SUMMARY OF THE INVENTION The present invention provides a separate outer membrane vesicle (OMV), N. meningitidis (NV).
eisseria meningitidis) substantially pure class I outer membrane protein (OMP), a fragment of class I OMP and a continuous or discontinuous reactivity of bacterial antibodies to N. meningitidis, Class I OMP-derived oligopeptides containing immunogenic and protective epitopes and N. meningitidi
s) for isolated vaccination against OMVs, Neisseria meningitidis class I OMPs, fragments or oligopeptides.

Isolated OMVs, meningococcal class I OMPs, fragments or oligopeptides derived therefrom can be monovalent or multivalent subunit vaccines alone, in mixtures, or in chemical conjugates or gene fusions. Can be used as a body. In a preferred vaccine, different epidemiologically related Neisseria meningitidis strains are used. In addition, a separate OM
V, class I OMPs, fragments or oligopeptides can be used in combination (as mixtures, fusions or conjugates) with other N. meningitidis antigens. For example, they are N. meningitidis capsular polymers or oligomers (or fragments thereof).
Or in combination with different subtypes of class I outer membrane proteins (or their epitopes). In addition, they can be used with other infectious bacterial, viral, fungal or parasitic antigens. Class I OMP T
Cellular epitopes have also been defined and these can be used in combination with other vaccine components to enhance the protective immune response to the vaccine.

The present invention relates to methods for producing isolated OMVs, class I OMPs, fragments and oligopeptides, and various vaccine formulations containing them. Isolated OMV class I OMPs can be produced by a mutant N. meningitidis strain that does not express class 2/3 outer membrane proteins. Fragments can be produced by cleavage of cyanogen bromide and subsequent purification. Separate OMV, Class I OMP,
Fragments and oligopeptides can be produced by recombinant DNA technology, chemical synthesis or chemical or enzymatic cleavage. These materials are subsequently added to the carrier peptide or protein by N. meningit.
idis) or other microbial antigens by chemical or genetic coupling to produce a multivalent antigen conjugate or fusion peptide or protein. They can be modified for conjugation, for example by the addition of amino acids or other coupling groups. For vaccination,
Separated OMVs, class I OMPs, fragments or oligopeptides can be formulated in any of the described forms, optionally with additives, eg adjuvants, in a pharmaceutically acceptable excipient.

The invention also relates to an isolated nucleic acid encoding a Class I OMP, fragment or oligopeptide. The nucleic acid can be incorporated into a suitable expression system which produces a discrete OMV, class I OMP, fragment or any oligopeptide derived therefrom. These nucleic acids can be modified as fusions of genes to contain additional polypeptide-encoding sequences useful in enhancing the immune response to vaccine formulations containing the expressed fusion polypeptides. it can. In addition, N. meningitidis class I OMPs are homologous in amino acid sequence and structure to other gram-negative pathogen porin proteins, thus providing class I OMPs of the invention. , Fragments and oligopeptides allow the development of vaccines for other Gram-negative pathogens.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. Overview of amplification of genes encoding Neisseria meningitidis class I outer membrane proteins by PCR (polymerase chain reaction).

Figure 2. A 5'gene sequence encoding VR1 (first variable region) of an outer membrane protein of class I of several N. meningitidis subtypes.

FIG. 3'gene sequence encoding VR2 (second variable region) of class I outer membrane protein of several N. meningitidis subtypes.

FIG. Scanning of epitopes by reaction of solid decapeptides with monoclonal antibodies that span the predicted amino acid sequences of class I proteins from strains P1.7.16, P1.16 and P1.15. Adjacent decapeptides differ by 5 amino acid residues. The annotations indicate the specificity of the strain from which the sequence was derived, the mAB used and its subtype.

FIG. Reaction of a series of overlapping decapeptides corresponding to the variable regions VR1 and VR2 with a monoclonal antibody, adjacent peptides differ by a single amino acid residue. The annotations indicate the specificity of the strain from which the sequence was derived, the mAB used and its subtype.

FIG. N. meningitidis Class I OM
Construction of recombinant flagellin expressing an epitope of the variable region of P subtype P1.7.16.

FIG. 7. N. meningitidis Class I OM
Structure of recombinant flagellin expressing a variable region epitope of P subtype P1.7.16.

FIG. Typical high performance liquid chromatography chromatogram of recombinant flagellin.

FIG. 9. Representative analysis of recombinant flagellin by SDS-PAGE.

Figure 10. N. meningitidi (M. meningitidi) joined to CRM ₁₉₇
Representative western blot analysis of conjugates consisting of epitopes of class I OMPs of s).

Figure 11. N. meningitidis Class I OM
Putative conformation of P subtype P1.7.16.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides isolated OMVs, N. meningitidis class I OMPs,
Vaccine consisting of a fragment of OPM (eg, prepared by application of cyanogen bromide) and an oligopeptide bearing an epitope of the outer membrane protein; isolated OMV using a mutant strain that does not express the outer membrane protein of class 2/3 ,
Preparation of pure class I outer membrane proteins; recombinant DNA
It relates to the preparation of pure class I outer membrane proteins over the minutes separated with the aid of cloned DNA in an expression vector. The present invention also provides a separate OMV, class I.
Of OMPs or parts thereof, fusions of genes of class I OMPs, genetic engineering applications for the purpose of producing proteins or epitopes thereof; and epitopes with short peptides can be prepared synthetically, It relates to the preparation of multivalent class I outer membrane vaccines.

The Neisseria meningitidis class I outer membrane proteins include strains A, B, C, W containing epitopes of the appropriate subtype.
It was shown to elicit a strong bacterial immune response against the strains, whether from the -135 and Y strains. Polysaccharide vaccines can be augmented or replaced by the vaccines according to the invention as broad-spectrum vaccines against most serotypes. Protective Bacterial Monoclonal Antibodies Specific for Class I Outer Membrane Proteins React Strongly with Cleaved Fragments and Short Synthetic Peptides Prepared Using the Amino Acid Sequence of Class I Outer Membrane Proteins .
Since the meningococcal illness is now mainly caused by group B Neisseria meningitidis, and the class I outer membrane proteins present in group B Neisseria meningitidis are also group A, C, W −13
5, because it is present in Y meningococci, N. menin
Vaccines of the invention consisting of one or more class I OMPs derived from group B of Gitidis) are group A, C, W-1.
It should be effective in preventing diseases caused by 35 and Y. Preferably, the preparation of such a vaccine starts with at least two different immunogenic and protective epitopes, selected on an epidemiological basis. Vaccines according to the invention are prepared, for example, by fragmenting at least one protein, or cyanogen bromide, obtained in an OMV formulation or by purification from a mutant strain producing one or more calf intestinal phosphatases. At least two fragments, or about 10 major epitopes, or at least two synthetic peptides, selected from the products, obtained by expression of the gene via recombinant DNA technology and containing the desired epitope. The greater the number of different protective epitopes, the better the vaccine, in order to maximize efficacy against a broad range of Neisseria meningitidis strains. In addition, the vaccine according to the invention may advantageously contain Neisseria meningitidis A and C or optionally W-135 and Y polysaccharides and / or detergents. Preferably, the A and C polysaccharides are covalently coupled to a protein or polypeptide carrier. These carriers are
For example, include: Separate OMV, Class I
OMP proteins, non-toxic mutations (CRM), recombinant Salmonella flagellin and viral particles, eg retroviral VP6 protein,
VR1 and VR2 of hepatitis B surface antigen or retrovirus
Protein. Detergents that generate both zwitterions, cations, anions and non-ions can be used. An example of such a detergent is the Zwittergent (Zw
ittergent) 3-10, Zwittergen
t) 3-14 (N-tetradecyl-N, N-dimethyl-3-ammonia-1-propanesulfonate), Tween (Tw
een) -20, sodium deoxychlorate, sodium chlorate and octyl glucoside. The vaccine according to the invention may also contain an absorbent, for example aluminum hydroxide, calcium phosphate; or advantageously aluminum phosphate. Fragments, proteins, peptides can also be immunostimulatory complex (ISCOM) for release.
S), can be treated with liposomes or microspheres and / or can be used as an adjuvant or in combination with other adjuvants, thus giving greater immunogenicity.

The present invention includes isolated OMVs, substantially pure Neisseria meningitidis class I outer membrane proteins (of any subtype) and fragments of proteins containing epitopes thereof. The fragment can be a portion with a molecular weight of 25 kD or less, containing an epitope bounded by antibodies of protective bacteria against N. meningitidis. These include proteolytic fragments and synthetic oligopeptides, which are composed at least in part of an amino acid sequence corresponding to an epitope of class I OMP.

The isolated OMVs, class I OMPs, fragments or epitope-containing oligopeptides derived therefrom can consist of amino acid sequences that differ from the native sequence but are essentially bioequivalent. These sequences include those sequences in which a functionally equivalent amino acid residue is replaced with a residue within the sequence resulting in a silent change. For example, one or more amino acid residues in the sequence can be replaced by another amino acid of similar polarity, which acts as a functional equivalent and results in a silent change. Substituents for amino acids in a sequence can be selected from other members of the class to which the amino acid belongs. For example, non-polar (hydrophobic) amino acids include glycine, alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

In addition, the isolated OMVs, class I OMPs, fragments or oligopeptides may contain other classes of class I OMPs of different subtypes, T cell epitopes, B cell epitopes, carrier peptides or proteins or adjuvant molecules. It can be modified for conjugation to other molecules it includes (eg, by attachment of coupling groups, eg, cysteine and / or lysine of amino acids or other linking groups and / or spacer groups).

As described in detail below, class I OMPs, fragments or oligopeptides are used in vaccines in different forms (eg, alone, in a mixture, or as gene fusions produced from conjugates and recombinant DNA vectors). can do. For these purifications, the material can be produced by isolation from N. meningitidis, by proteolytic digestion, by chemical synthesis or by expression as a recombinant molecule.
Separated OMVs, Class I OMPs and fragments and Class I
Methods and uses for the production of OMP oligopeptides are described below.

Modeling and structural analysis of class I OMP proteins was performed using principles for several E. coli outer membrane proteins. [Vogel, H.
Et al., Journal of Molecular Biology (J. Mol. Biol.), 190 : 191 (1986);
nce), T. et al., Journal of Molecular Biology (J. Mol. Biol.), 201 , 493 (1988) and Tommassen, J., "Membrane Biogenesis", NATO ASI Series (Series) H16, pp351, Springer-Verlag, NY (1988)]. The derived amino acid sequences of Class I OMPs were used for modeling studies and comparisons. Amino acid sequence homology was compared to other Gram-negative bacteria and similarities were established for protein structure. The exposed surface loops and transmembrane structure were very similar to these proteins.
Pathogenic Gram-negative to be evaluated and included in vaccines based on amino acid sequence using information about N. meningitidis variability and protective epitopes in certain regions and One can predict where the protective epitopes for bacteria will be.

Separated OMV production OMVs are described by Beuvery et al. (1983), Infection.Immun. 40 :
After fragmentation as described in 369-380, it can be produced from culture supernatants or bacterial cells. 1
OMVs with more proteins from N. meningitidis can be isolated from strains engineered to express heterologous proteins.

Production and Purification of Class I OMPs and CNBr Fragments Class I and Class 3 outer membrane proteins have been described by Beuvery EC et al.
tonie wan Leeuvenhoek J. Microbiol.) 52 : 232 (198
It can be separated as described in 6).
The production of substantially pure class I OMPs free of class 2 or 3 OMPs is achieved by this method in class 2/3 OM.
This is accomplished using a mutant N. meningitidis strain that does not express P. A preferred strain for the production of class I OMP is C
It is HIII5 which has been entrusted as BS 636.89.

The fragments were brominated as described by Teerlink T. et al., Journal of Experimental Medicine (J.Exp.Med.), 166 : 63 (1987) for N. gonorrhoeae proteins. It can be produced by a cyan fragment. The N-terminal fragment is called CB-1 and the C-terminal fragment is called CB-2. These CNBr fragments are
Vydax ^R or Aquapor ^R
It can be purified via reverse phase HPLC on an R-300 column using a water / acetonitrile gradient. Alternatively, the fragments can be purified by multiple cold precipitations of trichloroacetic acid. These procedures involve disturbing pollutants (eg,
> 95% of buffer salts, detergents and fragment contaminants).

Preparation of Fragments and Oligopeptides Containing Class I OMP Epitopes A. Preparation of Proteolytic Digestion Oligopeptides containing epitopes reactive with bacterial antibodies against N. meningitidis are:
Fragments of class I OMP, CB-1 or CB-2 can be produced by digestion with proteases such as endo-Lys-C, endo-Glu-C and staphylococcins noV8-protease. The digested fragments can be analyzed, for example, by high performance liquid chromatography (HPL
C) can be purified by technology.

B. Preparation by Chemical Synthesis Oligopeptides of the invention are prepared by standard solid phase peptide synthesis [Barany, G. and Merrfield (Merrfield.
ifield), RB, The Peptides 2 : 1
−284, Gross, E. and Meienhofer, J. Ed., Academic Press (Academ)
ic Press), New York], t-butyloxycarbonyl amino acid and phenylacetamidomethyl resin [Mitchell, AR et al., Journal of Organic Chemistry (J.Org.Chem.) 4
3 : 2845-2852 (1978)] or 9 on a polyamide support.
-Fluorenylmethyloxycarbonyl amino acid [dryland, A. and Shepard (Sheppar
d), RC, Journal of Chemical Society (J. Chem. Soc.) Perkin Trans. I, 125-137 (198
6)] can be used for synthesis. Alternatively, synthetic peptides can be synthesized by pepscan [Geisen (G
eysen), HM et al., Journal of Immunological Methods (J.Immunol.Methods) 03: 259 (1987): Proceedings of National Academy of Sciences (Proc.Natl.Acad.Sci.) USA, 81 : 399
8 (1984)], Cambridge Research Biochemicals, Cambridge, England, or by standard solution phase peptide synthesis. Amino acid deletions or substitutions (and including extensions and additions to amino acids) in other ways that do not substantially reduce the immunological properties of the oligopeptide.

C. Preparation by Recombinant DNA Technology Class I OMPs, and fragments and oligopeptides displaying epitopes of Class I OMPs can be produced by recombinant DNA technology. In general, these are the desired
OMP, [Barlow et al. (1989) Molecular Microbiology (Mol. Micro.), 3 : 131] Obtaining a DNA sequence encoding the sequence of the fragment or oligopeptide, and of a suitable vector / host It involves introducing into the expression system one or more similar or different DNA sequences of a class I OMP and expressing it there. The DNA can consist of a gene encoding a class I OMP or a segment of a gene encoding a functional epitope of OMP. DNA is N. meningitidis (Nm
eningitidis) to other antigens (eg, the same or different classes of outer membrane proteins), or to other bacterial, viral, parasitic or fungal antigen-encoding DNAs to form genetically fused ( Multivalent antigens (that share a common peptide backbone) can be made. For example, a fragment of Class I OMP is used by N. meningitidis (N.
ingitidis) to other class I outer membrane proteins (or fragments or epitopes thereof) of different subtypes to produce fusion proteins consisting of a stable antigenic determinant of multiple class I outer membrane proteins. You can

Genetic engineering techniques can also be used to characterize, modify and / or adapt the encoded peptide or protein. For example, site-directed mutagenesis can modify OMP fragments in regions outside the protective domain, eg, to increase solubility of subfragments to facilitate purification. DNA is also
It can be engineered to overproduce or combine OMP fragments in different organisms.

DNA encoding class I OMPs, fragments or oligopeptides has been described by Barlow, AK et al., Infect. Immun. 5
5 : 2734-40 (1987) and [Barlow et al., Molecular Microbiology, Mol. Micro.
3 : 131 (1989)] and can be synthesized or separated and sequenced. Class I OM
The gene for P is derived from bacterial DNA and is Mullis.
And Faloona, (1987) Method.Enzym.
155: 335-350 can be amplified using the primer sequences disclosed therein. Relevant DNA sequences for different subtypes of class I OMPs can be obtained and the amino acid sequence deduced by the procedures described.

A variety of host-vector systems can be used to express the oligopeptides of the invention. Primarily, the vector system must be compatible with the host cell used. Host-vector systems include, but are not limited to: bacteriophage DNA, plasmid D.
Bacteria transformed with NA or cosmid DNA; microorganisms such as yeast containing the yeast vector system;
Mammalian cell lines infected with viruses (eg, vaccinia virus, adenovirus, etc.); Insect cell lines infected with viruses (eg, baculovirus). The expression elements of these vector systems vary in their strength and specificity. Depending on the host-vector system utilized,
Any of a number of suitable transcription and translation elements can be used.

To obtain efficient expression of cloned DNA,
The promoter must be present in the expression system. RN
A polymerase normally binds to a promoter and initiates transcription of a gene or group of linked genes and regulatory elements (called operons). Promoters change their “strength”, ie their ability to promote transcription. It is desirable to have high levels of transcription using a strong promoter and therefore high levels of expression of DNA. Depending on the host cell system, any one of the suitable promoters can be used. For example, E.
coli, its bacteriophage or plasmid, promoters such as the lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, and coliphage amda P _R or P _L promoter, and Other non-limiting: lacUV5, ompF, bla, lpp, etc. can be used to direct high level transcription of adjacent DNA segments. In addition, the hybrid trp-lacUV
The inserted DNA can be transcribed using the 5 (tac) promoter or other synthetic DNA technology.

Bacterial host cells and expression vectors can be chosen which do not inhibit the action of the promoter unless specifically induced. In some operons,
Addition of a specific inducer is necessary for efficient transcription of the inserted DNA; for example, the lac operon is either lactose or IPTG (isopropyl othio-beta-D).
-Galactoside). Various operons, such as trp, are under different control. When tryptophan is absent in the growth medium, trp operon is induced; and lambda P _L promoter, host cells containing a temperature sensitive lambda receptor, for example, be induced by increasing the temperature in the cI857 it can. In this way, more than 95% of promoter-directed transcription can be inhibited in uninduced cells. In this way, expression of the recombinant peptide or protein can be controlled. this is,
Whether the expression product of the DNA is lethal to the host cell,
Or if it is harmful, it is important. In such cases, the transformants can be cultured under conditions such that the promoter is not induced; then, when the cells reach the appropriate density in growth medium, the promoter is induced for protein production. can do.

One such promoter / operator system is
The "tac" or trp-lac promoter / operator system [Russell and Bennett
t), 1982, Gene 20: 2312-243; DeBo
er), European Patent Application No. 67,540, May 18, 1982]. The promoter of this hybrid is -35bo (-35 region)
And the lac promoter of the −10 bp [−10 region or Pribnow box] (the sequence of DNA that is the RNA polymerase binding site). In addition to maintaining the strong promoter properties of the tryptophan promoter, tac is also controlled by the lac repressor.

Enhancer sequences (eg, 72 bp tandem repeats of SV40 DNA or retroviral long terminal repeats or LTRs) when cloned in eukaryotic host cells
Can be inserted to increase transcription efficiency. Enhancer sequences are a set of eukaryotic DNA elements that appear to increase the efficiency of transcription in a manner that is relatively independent of their location and orientation with respect to nearby genes. A classical promoter element that must be located immediately 5'to the gene [eg, polymerase binding site and Goldberg-Hognes (Goldbe
rg-Hogness) "TATA" box], the enhancer sequence has a prominent function of functioning upstream from, within, or downstream of eukaryotic genes; Position is not so critical.

Specific initiation signals are also required for efficient gene transcription and translation in prokaryotic cells. These transcriptional and translational initiation signals may vary in "strength" as measured by the amount of gene-specific messenger RNA and protein synthesized, respectively. A DNA expression vector containing a promoter may also contain any combination of various "strong" transcription and / or translation initiation signals. For example, efficient translation in E. coli requires approximately 7-9 relative to the initiation codon (ATG) to provide a ribosome binding site.
It requires a Shine-Dalgarno sequence at base 5 '. Thus, SD-ATG combinations that can be utilized by the ribosome of the host cell can be used. Such combinations include but are not limited to: coliphage lambda cr
SD-AT from o gene or n gene or from E. coli tryptophan E, D, C, B or A gene
G combination. In addition, produced by recombinant DNA
Other techniques can be used, including SD-ATG combinations or the incorporation of synthetic nucleotides.

Any of the methods described for the insertion of DNA into expression vectors can be used to join promoters and control elements of other genes into specific sites within the vector. N. mening for expression
Itidis) sequences can be inserted at specific sites in the expression vector with respect to the vector's promoter and control elements, thus allowing the foreign gene sequences to be expressed by the host cell when the reduced DNA molecule is introduced into the host cell. (Ie, transcribed and translated).

The recombinant DNA vector can be introduced into an appropriate host cell (bacteria, virus, yeast, mammalian cell, etc.) by transformation, transduction or transfection (depending on the vector / host cell system). A host cell containing the vector may be one or more suitable genetic markers that normally depend on the vector, such as ampicillin resistance or tetracycline resistance in pBR322, or thymidine kinase activity in a eukaryotic host system. Selection based on the expression of Expression vector The cloning vector can be derived from the cloning vector, which contains the functioning marker.
Such cloning vectors can include, but are not limited to: SV40 and adenoviruses, vaccinia virus vectors,
Insect viruses such as baculovirus, yeast vectors, bacteriophage vectors such as lambda gt-WES-lambda B, sharon 28, sharon 4A, lambda 4A, lambda gt-1-lambda BC, lambda gt-1. -Lambda B, M13mp7, M13mp8, M13mp9, or plasmid DNA vectors such as pBR322, pAC105, pVA51, pACYC177, p
KH47, pACYC184, pUB110, pMB9, pBR325, ColE1, pSC10
1, pBR313, pML21, RSF2124, pCR1, RP4, pBR328 etc.

Expression vectors containing DNA inserts can be identified by three general approaches: (1) DNA-DNA consisting of sequences homologous to the inserted gene.
(2) presence or absence of "marker" gene function (eg, resistance to antibiotics, transforming phenotype, thymidine kinase activity, etc.);
And (3) expression of the inserted sequences based on the physiological immunological or functional properties of the gene product.

Once a putative recombinant clone expressing the desired Class I OMP amino acid sequence is identified, the gene product can be analyzed as follows. Immunological analysis is of essential importance. This is because the ultimate goal is to use the gene product as an antigen in vaccine formulations and / or in diagnostic immunoassays. The expressed peptide or protein should be immunoreactive with bacterial antibodies to N. meningitidis. This reactivity can be demonstrated by standard immunological techniques such as radioimmunoprecipitation, radioimmuno competition, ELISA or immunoblot.

Once the gene product has been identified as an oligopeptide containing a fragment of class I OMP or a functional epitope thereof, it is subjected to standard methods, eg chromatography (eg ion exchange, affinity and sizing columns). Separation and purification by chromatography), centrifugation, differential solubility, or by purification or any other standard technique for proteins. There are several techniques for purification of heterologous proteins from prokaryotic cells. See, eg, Olson, US Pat. No. 4,518,526, Wetz.
el), US Pat. No. 4,599,197 and Hung.
Et al., U.S. Pat. No. 4,734,362. However, the produced and purified preparation should be substantially free of host toxins that can be harmful to humans. In particular, the purified peptide or protein should be substantially free of endotoxin contaminants when expressed in Gram-negative bacterial host cells such as E. coli or Salmonella.

The Class I OMPs, fragments and oligopeptides of the invention can be formulated as monovalent or multivalent vaccines. These materials can be used as produced or separated by the methods described above. They can be mixed, conjugated or fused with other antigens, eg B cell or T cell epitopes of other antigens. further,
They can be conjugated to carrier proteins as described below for oligopeptides.

When using the hapten oligopeptide (ie, a peptide that reacts with a cognate antibody but is unable to elicit an immune response by itself), it can be conjugated to an immunogenic carrier molecule. Conjugation to an immunogenic carrier renders the oligopeptide immunogenic. Bonding can be done by standard procedures. Preferred carrier proteins for hapten oligopeptides include toxins, toxoids, or tetanus, diphtheria, pertussis, Pseudomonas, E. coli, Staphylococcus, and streptococcus (St.
reptococcus) is a mutant cross-reactive substance (CRM). A particularly preferred carrier was derived from P. diphtheriae strain C7 (β197) which produces the CRM ₁₉₇ protein,
It is diphtheria toxin XRM ₁₉₇ . This strain has ATCC Accession No. 53281. Alternatively, carrier proteins or fragments or epitopes of other immunogenic proteins can be used. For example, haptens can be coupled to bacterial toxins, toxoids or T cell epitopes of CRM. See, US Patent Application No. 150,688, filed February 1, 1988, entitled "Synthetic peptides representing T cell epitopes as carrier molecules for conjugate vaccines", the teachings of which are incorporated herein by reference. Other carriers are rotavirus VP
6, hepatitis B surface antigen or parvovirus VP1 and VP2
Includes.

The peptide or protein of the present invention can be used for N. meningitidis (N.
meningitidis) or other organism antigens and can be administered as a multivalent subunit vaccine. Some of the other organisms include: H. influenzae, N. meningitidis, B. catarrhalis, N. gonorrhea.
oeae), E. coli, Streptococcus pneumoniae (S. pneumoniae), etc.
For example, they can be administered in combination with a N. meningitidis oligosaccharide or polysaccharide capsular component. Capsular components are a group of serology, such as
It can be derived from any of A, B, C, D, X, Y, Z, 29E and W135.

Different subtypes of class I outer membrane proteins can be used. These can be used in combination to elicit bacterial antibodies against N. meningitidis. For example, fragments derived from class I outer membrane proteins of the P1.7.16 subtype can be labeled with multiple subtypes, eg, P1.1, P1.1, 16; P1.2; P1.6; P1.9; P
1.15; P.16; or P1.4 [Abdillahi,
H. et al., 1988 Microvial Pathogenesis (Micro.
Pathog), 4:. 27] can be used together with the fragments of the outer membrane protein or outer membrane proteins, or the form or chemical conjugates of polysaccharides and mixtures meningococcal of. For administration in combination with epitopes of other outer membrane proteins, they can be administered separately, as a mixture or as a fusion peptide or protein of a conjugate or gene. Conjugates can be formed by standard techniques for coupling protein materials or by coupling saccharide polymers to proteins. The fusion, as described,
It can be expressed from fused gene constructs prepared by recombinant DNA technology.

As mentioned above, class I OMPs, fragments or any oligopeptides derived from them can be used for other pathogenic organisms [eg bacteria (microencapsulated or non-microencapsulated), viruses, fungi and parasites].
It can be used in combination with the antigen. Additional examples of other organisms include RS virus, rotavirus, malaria parasite, and Cryptococcus neoformans.

In formulating a vaccine composition having peptides or proteins alone or in various combinations as described, the immunogen is adjusted to the appropriate concentration and formulated using any suitable vaccine adjuvant. Suitable adjuvants include, but are not limited to: surface-active substances such as hexadecylamine,
Octadecylamine, octadecyl amino acid ester,
Lysolecithin, dimethyldioctadecyl ammonium bromide, methoxyhexadecyl glycerol, and pluronic polyols; polyamines such as pyran,
Dextrasulfate, poly IC, carbopol; peptides such as muramyl peptides and derivatives, dimethylglycine, tuftsin; oil emulsions; and mineral gels
For example, aluminum hydroxide, aluminum phosphate, etc., lymphokines and immunostimulatory complexes (ISCOMS).
Immunogens can also be incorporated into liposomes, microspheres, or conjugated to polysaccharides and / or other polymers used in vaccine formulations.

The vaccine can be administered to humans or animals in various ways. These include intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral and intranasal routes of administration.

Live Vaccine The peptides and proteins of the invention can be administered as a live vaccine. Vaccine recipients are inoculated with recombinant microorganisms that grow in the recipients, express class I OMPs, fragments or oligopeptides, and elicit an immune response against N. meningitidis . Live vaccines include: adenovirus, cytomegalovirus and preferably poxviruses such as vaccinia [Paoletti and Panicali, US Pat. No. 4,603,112] and attenuated Salmonella (Sal).
monella) stock [Stocker, US Pat.
0,081 and Curtiss et al., Vaccine (Vacc
ine) 6 : 155-160 (1988)]. In addition, Class I OMP
Can be incorporated into the flagella of an attenuated bacterial strain.

Live vaccines are particularly advantageous. Because they lead to prolonged stimulation, which can confer substantially long-lasting immunity. When the immune response is protective against subsequent N. meningitidis infection, the live vaccine itself is N. meningitidis
Can be used in a prophylactic vaccine against

Multivalent live vaccines should be prepared from one or several recombinant microorganisms expressing different N. meningitidis epitopes (eg outer membrane proteins from other subtypes or epitopes) You can In addition, epitopes for other pathogenic microorganisms can be incorporated into the vaccine. For example, the vaccinia virus can be engineered to contain coding sequences for other epitopes in addition to that of N. meningitidis. Such recombinant virus itself can be used as an immunogen in a multivalent vaccine. Alternatively, formulating as a multivalent vaccine a mixture of different genes, each specifying a different epitope of the outer membrane protein of N. meningitidis and / or the epitope of another disease-causing microorganism. You can

Inactivated virus vaccines can be prepared. Inactivated vaccines are usually "killed", ie, infectious, destroyed by chemical treatment (eg, formaldehyde). Ideally, the infectivity of the virus is destroyed without affecting the immunogenic proteins of the virus. To prepare the inactivated vaccine protein, the recombinant virus expressing the desired epitope is grown in culture in large quantities to provide the required amount of the relevant antigen. A mixture of activated viruses expressing different epitopes can be used for the formulation of "multivalent" vaccines. In some cases, these "multivalent" inactivated vaccines are preferably live vaccine formulations. Because, potential difficulties arise from the mutual interference of live vaccines given together. In any case,
The inactivated virus or mixture of viruses can be formulated in a suitable adjuvant to enhance the immune response to the antigen. Suitable adjuvants include the following: surface-active substances such as hexadecylamine, octadecyl amino acid esters, octadecylamine, lysolecithin, dimethyl-dioctadecyl ammonium bromide, N, N-dicoctadecyl-N ', N'-bis. (2
-Hydroxyethyl-propanediamine), methoxyhexadecylglycerol, and pronpolyols; polyamines such as pyran, dextransulfate, polyIC, carbopol; peptides such as muramyl peptide and its derivatives, dimethylglycine, tufticine; oily milk Suspensions; and mineral gels such as aluminum hydroxide, aluminum phosphate, and lymphokines.

Illustrative Example 1: Monoclonal Antibodies to Class I OMPs and Their Activity Type-specific monoclonal antibodies to various meningococcal class I outer membrane proteins were prepared. These monoclonal antibodies recognize the following subtypes: P1.
1; P1.2; P1.6; P1.7; P1.9; P1.10; P1.15; P.16; and P1.17
(Here we call P1.14). Monoclonal antibody is RIV
M, Biltuben, Netherlands, "Mononal Kit Seroty Monoclonal Kit for Serotyping Meningococci.
ping Meningococci) ”. All these monoclonal antibodies react with SDS (sodium dodecyl sulfate) denatured protein when tested by Western blotting. It was also shown that a number of these monoclonal antibodies react with the 25 kD CNBr fragment of the 42 kD class I outer membrane protein. As this result implies, the epitopes of class I outer membrane proteins are predominantly of linear type and can therefore be copied with synthetic peptides. The epidemiological results of the tests carried out by the Applicant show that the described monoclonal antibodies are capable of subtyping the majority of Neisseria meningitidis of Groups A, B, C, which is of limited heterogeneity. Suggest. Each class I outer membrane protein also appears to contain two individual type-specific epitopes [Abdillahi and Poolman, Microvial Pathog., 1988. , 4 : pp.27-32; ibid, FEMS
Microbiology and Immunology (Micorobiol.Imm
unol.) 47: pp.139-144].

Purified class I outer membrane protein (see below) subtype P1.7.16, derived from a culture of the class 2 / 3-free mutation (HIII5), was 1:64 in mice at a dose of 2.5 μg. Dilutions of sera appeared to elicit a bacterial antibody response. Monoclonal antibodies against meningococcal class I outer membrane proteins, class 2/3 outer membrane proteins and lipopolysaccharides were compared for bacterial action. Monoclonal antibodies against class I outer membrane proteins appeared to have the strongest bacteria (see Table 1). Bactericidal response is described by Poolman, JT (1985), Schoennick (S
choolnik, GK et al. (ed.), "Pathogenic Neisseria genus" (Pat
hogenic Neisseriae) "ASN Publication (Public
ation), Washington DC, p.562.

The bactericidal activity of these monoclonal antibodies correlates well with in vivo protective activity measured in a model of rat meningitis in Saukkonen et al., 1987, Microbial Pathoggen. 3 : 261. Seems to have a relationship.

Example 1A: Construction of a Neisseria meningitidis strain with multiple class I genes Substitution of chromosomal genes by clones, a slightly different variant, was described for Neisseria gonorrhoeae. [Stein, DC, Clin.Microbio
Rev. 2 (Suppl.), S146-S149 (1989)]. We have found that this method can be applied to class I genes in Neisseria meningitidis. This was done in the following way: (i) The class I genes of strain 2996 (subtype P1.2) were cloned into the vector pTZ19R. [Me
ad), DA et al., Protein Engineering (Protei
n Engineering) 1 , 67 (1986)]. The complete gene is
It is located on the 2.2 kb Xba I fragment bound to the Xba I digested vector DNA.

(Ii) The resulting plasmid was strain H44-76 (subtype P1.7.
It was used for the transformation of 16). Acceptor cell lines were incubated with plasmid DNA in the presence of Mg ²⁺ and normal Neisseria meningitidis medium; they were subsequently diluted and plated, and the resulting colonies were P1.2-specific monoclonal. Tested for their ability to bind the antibody. Such transformants were found at a frequency of approximately 10 ^-3 . Further characterization showed that substitution of the H44 / 76 class I gene did occur. The essential feature of this method is that N.
Ningitidis) is the presence of a donor gene on a circular plasmid DNA molecule that is unable to replicate. This is because the use of linearized DNA did not produce any transformants.

Construction of strains with two class I genes was performed by modification of the method described above. For this purpose, the P1.2 class I gene was inserted into the clone class 5 gene.
The family of Class 5 genes has two characteristics that make it particularly suitable for this organization.
[Meyer, TF and Van Putten (Van
Putten), JPM, Clin.Microbiol.Rev. 2 (Supp
l.), S139-S145 (1989): (i) there are 4 or 5 class 5 genes in the genome of Neisseria meningitidis, and (ii) expression of these genes under laboratory conditions. Growth is unnecessary in. The class 5 gene was cloned from the H44 / 76 strain and the P1.2 gene was inserted into the class 5 gene or into the Sph I site located very closely thereto. The resulting hybrid plasmid,
pMC22 was used to transform strain HIII5, a class 3 deletion mutation of H44.76. Colonies that reacted with the P1.2-specific monoclonal antibody were isolated and characterized. 9 out of 10 such transformants were of the acceptor strain
It was discovered that the P1.16 epitope has been lost. As this shows, all these classes of recombination occur only between class I genes, resulting in subtype substitutions. However, one transformant was discovered that made both class I subtypes, P1.7.16 and p1.2, resulting in recombination between the sequences of the class 5 genes on the plasmid and chromosome. Suggest that it is not. This was confirmed by Western and Southern blotting, where Western blotting revealed the presence of both types of class I proteins, and Southern blotting demonstrated the acquisition of a second class I gene.

By continuing this construction with other class I subtypes, strains with 4 or 5 different class I genes can be created. The same class 5 genes can be used in each subsequent transformation step, different class 5 genes can be cloned separately,
And can be used. These recombinant strains can be used to prepare mixtures of different purified Class I OMPs.

Example 1B: Purification of isolated OMVs from bacteriological culture Purification was performed by Beuvery et al. (1983) Infect. Immun. 40 :
Carry out according to 369-380.

This culture can be carried out using the desired wild type strain, a mutant Neisseria meningitidis strain lacking the class 2/3 outer membrane protein and / or a homologous and heterologous recombinant microorganism. Since the microorganism expresses one or more desired Neisseria meningitidis class I outer membrane proteins and / or epitopes by overproducing the vector with or without an existing open reading frame, Fusion proteins or proteins with exchanged epitopes can be prepared.

The readily available wild-type strains are: H44 / 76 (B: 15: P1, 7.16) [Holten E.,
Norway, acceptance number CBS635-89]; 187 (B: 4: P
1.7) [Etienne J., France]; M108
0 (B: 1: P1, 1.7) [Frasch, USA]; Swis
s4 (B: 4: P1, 15) [Hirschel B., Switzerland]; B2106I (B: 4: P1, 2) [Berger U.,
West Germany]; 395 (B: NT: P1, 9) [Jonsdottir K., Iceland]; M990 (B: 6: P1,
6) [Frasch C., USA]; 2996 (B: 2b: P
1, 2) RIVM, Netherlands; M982 (B: 9: P1, 9) [Frasch C., USA]; H355 (B: 15: P1, 15)
[Holten E., Norway]; 6557 (B: 1
7: P1, 17) [Zollinger W., USA] and B40 (A: 4: P1, 10) [Achtman M., West Germany]. One example of a class 3 negative mutation is HIII5
(B:-: P1.16) Accession number is CBS636.89.

These strains were inoculated from pre-culture into shake flasks at -70 ° C and from them 40, 150 or 3
Transferred into 50 liters of fermentation culture. The semi-synthetic medium had the following composition: L-glutamic acid 1.3 g / l, L-cysteine.HCl 0.02 g / l, Na ₂ HPO ₄ 2H ₂ O 10 g / l, KCl
0.09g / l, NaCl 6g / l , NH 4 Cl 1.25g / l, MgSO 4 · 7H 2 O
0.6 g / l, glucose 5 g / l, Fe (NO ₃ ) ₃ 100 μmol,
Yeast dialysate.

During culture in the fermenter, and monitoring the pH and PO _2, and adjusted automatically pH7.0~7.2 and air saturation 10%. Cells are grown to early stationary phase, harvested by centrifugation and washed with sterile 0.1 M NaCl, and -20 ° C.
Stored at or lyophilized.

Example 2: Purification of Class I Outer Membrane Proteins from Bacteriological Cultures This culture comprises the desired wild-type strain, a mutant Neisseria meningitidis strain that does not contain class 2/3 outer membrane proteins and / or homologues. Sex and heterologous recombinant microorganisms can be carried out by overproducing the vector with or without open reading frames and / or engineered reading frames present, or Since more than one desired Neisseria meningitidis class I outer membrane protein and / or epitope is expressed, fusion proteins or proteins with exchanged epitopes can be prepared.

The readily available wild-type strains are: H44 / 76 (B: 15: P1, 7.16) [Holten E.,
Norway, acceptance number CBS635-89]; 187 (B: 4: P
1.7) [Etienne J., France]; M108
0 (B: 1: P1, 1.7) [Frasch, USA]; Swis
s4 (B: 4: P1, 15) [Hirschel B., Switzerland]; B2106I (B: 4: P1, 2) [Berger U.,
West Germany]; 395 (B: NT: P1, 9) [Jonsdottir K., Iceland]; M990 (B: 6: P1,
6) [Frasch C., USA]; 2996 (B: 2b: P
1, 2) RIVM, Netherlands; M982 (B: 9: P1, 9) [Frasch C., USA]; H355 (B: 15: P1, 15)
[Holten E., Norway]; 6557 (B: 1
7: P1, 17) [Zollinger W., USA] and B40 (A: 4: P1, 10) [Achtman M., West Germany]. One example of a class 3 negative mutation is HIII5
(B:-: P1.16) Accession number is CBS636.89.

These strains were inoculated from pre-culture into shake flasks at -70 ° C and from them 40, 150 or 3
Transferred into 50 liters of fermentation culture. The semi-synthetic medium had the following composition: L-glutamic acid 1.3 g / l, L-cysteine.HCl 0.02 g / l, Na ₂ HPO ₄ 2H ₂ O 10 g / l, KCl
0.09g / l, NaCl 6g / l , NH 4 Cl 1.25g / l, MgSO 4 · 7H 2 O
0.6 g / l, glucose 5 g / l, Fe (NO ₃ ) ₃ 100 μmol,
Yeast dialysate.

During culture in the fermenter, and monitoring the pH and PO _2, and adjusted automatically pH7.0~7.2 and air saturation 10%. Cells are grown to early stationary phase, harvested by centrifugation and washed with sterile 0.1 M NaCl, and -20 ° C.
Stored at or lyophilized.

Bacterial mass, for example, 0.5 mol CaCl ₂ , 1%
(W / v) Zwittergent 3-14 (Zw
3-14) and 0.14 mol NaCl, pH 4.0 with the help of 10
Extraction was performed using 0 / g lyophilized bacterial mass. The suspension was stirred at room temperature for 1 hour and then centrifuged (1 hour, 3000 × g) after which the suspension was collected aseptically. 20% (v / v) ethanol was added to the supernatant and then stirred for 30 minutes and the product was centrifuged (30 minutes, 10,0
00xg), after which the suspension was collected aseptically. Then
The supernatant was concentrated by diafiltration in an Amicon Hollow Fiber System (HID x 50, cutoff 50,000) and CaCl ₂
And ethanol was removed. Concentrate 0.1 mol sodium acetate, 25 mmol EDTA, 0.05% Zw3-14, pH.
6.0 was diluted to the original volume and then concentrated by dilute filtration. This procedure was repeated 5 times again. The pH of the final concentrate
Adjusted to a value of 4.0. 20% (v / v) ethanol was added to the concentrate and after stirring for 30 minutes the product was centrifuged (30
Min, 10,000 xg). All proteins are purified by column chromatography in the presence of detergent, eg Zw3-14. Often apply gel filtration on Sepharose S-300 and ion exchange on DEAE Sepharose [Beuvery et al. (1986) s
upra]. The methods used, detergents, column chromatography are not the only applicable methods, and are examples and should not be considered as limiting.

Example 3: Preparation and Characterization of Peptide Fragments of Class I OMPs Cyanogen bromide was used to prepare Neisseria meningitidis Class I outer membrane proteins. Purified class I or class I
Or 3 outer membrane proteins were taken up in 70% (v / v) formic acid and treated with a 10-fold excess of CNBr for 16 hours at room temperature. CNBr and formic acid were removed by evaporation and replaced with 0.2M Tris HCl, 6M urea solution, pH 7.2. The supernatant was purified by gel filtration on Sephacryl S-2000. Beuvery et al. (1
986) supra.

Enzymatic digestion of the CB2 fragment To further delineate the epitope, the CB2 of N. meningitidis
The fragments were digested with endo-Arg-C, endo-Glu-C or V-8 and the resulting fragments were separated by HPLC. Briefly, in 1 ml of 25 mmol phosphate / 0.1 mmol Tris buffer (pH 8.0) containing 3 mol urea.
20 nmoles of CB2 fragment were added to 0.2
Nanomolar endo Arg-C (14-18 mg / ml in distilled water) or 0.22 nanomolar endo Glu-C (14-18 m in distilled water)
g / ml). The resulting digested fragments were separated by reverse phase HPLC using a Vydac-C4 column and a trifluoroacetic acid-acetonitrile gradient. End
The major peak eluted from the Arg-C digest had an apparent molecular weight of 7-9 Kdal, while the major peak observed after endo Glu-V-8 had an apparent molecular weight of 4-6 Kdal. The separated peaks were subsequently Western blotted to a pool of monoclonal antibodies [Adam].
I, 62-D12-8, MN5-C11G and MN14-C116).

The P1.16 epitope appears to reside on the C-terminal CNBr fragment of the class I outer membrane protein of H44 / 76 (B: 15: P1, 7.16). Further characterization of the P1, 16 epitope was performed by determining the amino acid sequence of 17Kd (N-terminal) and 25Kd (C-terminal). The C-terminal 25 Kd is further processed by VR protease, endo-LysC, endo-Glu-C and endo-Arg.
Fragmented with -C. The fragments that were positive with P1, 16 molar antibody were sequenced as much as possible. The sequence obtained is as follows: N-terminus of all proteins: N-terminus of the 25 Kd C-terminal CNBr fragment: Fragments reactive with the P1, 16 monoclonal antibody were separated using fragments of V8 protease and endo-Arg-C with 7-9 Kd and 4-6 Kd, respectively. The N-terminal Southern analysis here is as follows: Fragment of V8 7-9Kd: Arg-C 4-6Kd fragment Example 4: DNA sequence of the class I OMP gene The amino acid sequence of the class I OMP was deduced from the nucleotide sequences of the four meningococcal class I OMP structural genes of various subtypes. By comparison with the four amino acid sequences it was possible to predict the composition and position of these epitopes. Furthermore, the P1,7 and P1,16 epitopes were confirmed by peptide synthesis and demonstration of binding of the respective monoclonal antibodies.

Cloning of class I OMP into lambda gt11 [P
About 1 and 16, Beuvery et al. (1987) Infect.Immu
n.) 55 : 2743-2740] and subcloned in the M13 sequencing vector, and the DNA sequence determined by standard chain-terminating dideoxynucleotide techniques.

The complete derived amino acid sequences for the P1, 16; P1, 15, P1, 7.16 and P1,2 proteins are as follows: Example 5: DNA sequencing of class I OMP genes from different N. meningitidis serotypes Mullis and Faloona [Methods in Enzymol
ogy) 155: 335-50, 1987] polymerase chain reaction (PC
R) technology was used to amplify specific fragments of the entire class I OMP gene according to the scheme shown in FIG.

Apply primers to Applied Biosystems (Applie
d Biosystems) 380B DNA synthesizer and used in a standard PCR 30 cycle amplification reaction using Taq polymerase in a thermal cycler (Thermal Cycle).
r) [Perkin-Elmer Ce
tus), Norwalk, Connecticut] according to the supplier's recommendations. Amplified fragments of approximately 1300, 900 and 450 bp were generated from genomic DNA preparations of each serotype from the primer combinations shown in FIG. The primers used had the following sequences: 370A type automated DNA for "asymmetric PCR" amplification
Sequencer [Applied Biosystems (Applie
d Biosystems), California, California, USA)
An excess of single-stranded template for sequencing was synthesized using a 100x excess of primer with an 18 base extension at the 5'end. Taq polymerase was used in a standard dideoxynucleotide chain termination sequencing reaction with a PCR-generated fragment of the single-stranded class I gene as template.
Strains H44 / 76 (P1.7, 16), M1080 (P1.1, 7), H355 (P
1.15), 6940 (P1.6), 6557 (P1.14), 870227 (P1.1)
Sequences derived for the 0) and B40 (P1.10) gene fragments are shown in FIGS. 2 and 3.

Example 6: Confirmation of Amino Acid Sequences of Epitopes of Class I OMP Subtypes From these genes confirmed by direct sequencing of the Class I OMP genes, amino acids 24-34 and 176-187.
The sequence corresponding to was predicted to be significantly variable in the four class I OMP sequences. The N- or C-termini of the three amino acid sequences from these positions also indicate the stability of the epitope in the native protein sequence and a possible inclusion in these epitopes, which maximizes unexpected insertions or deletions. Was thought about. In addition, the DNA and amino acid sequences of other class I OMPs should be compared to the P1.7,16 sequence to allow maximum alignment and prediction of epitopes. The first variable region epitope and the second variable region epitope are referred to as VR1 and VR2, respectively. These regions encode subtype epitopes. This was confirmed by peptide synthesis and reaction of the peptides with p1.2; P1.7; P1.15 and P1.16 specific monoclonal antibodies.

A complete set of overlapping decapeptides that differ by 5 amino acids was prepared using the P1.16 protein sequence. Anti-P1.16 monoclonal antibody reacts as expected with the decapeptide YYTKDSTNNNL from P1.16
And did not react with other decapeptides.
(Fig. 4).

Strains H44 / 76 (P1.7, 16), M50 (P1.16) and MC51
(P1.15) Class I OMP regions 24-34 and 176-187
In 1 after (1,) an overlapping decapeptide having an amino acid sequence, more than one peptide reacted with a subtype-specific monoclonal antibody. In most cases, one or more groups of these overlapping peptides reacted with subtype-specific monoclonal antibodies more strongly than others (5th
Figure).

These peptides are designated as VR1 and VR2 epitopes. In the P1.7, 16 strain, the sequence YYTKNTNNNL is present and the change of D to N at residue 180 has some effect on reducing antibody binding. The sequence HYTRQNNTDVF in P1.15 is
It is in the same relative position in the protein as the P1.16 epitope and is responsible for binding to the anti-P1.15 monoclonal antibody. AQAANGGASG shows some binding, and 1-
A peptide 3 amino acids downstream shows even greater binding to the p1.7 monoclonal antibody. Array H of VR2
FVQQTPQSQP causes binding to anti-P1.2 monoclonal antibody. Sequence of proteins in P1.16 and P1.15 QPQVTN
GVQGN and PPSKSQP also appear to represent epitopes.

Example 6B: Identification of constant region epitopes of class I OMPs Surface loop-forming peptides were prepared and conjugated to tetanus toxoid. Biolyns
x) 4170 automated peptide synthesizer [Pharmasia (Pharm
acia) / LKB] was used for continuous flow solid phase synthesis with the following exceptions. In the last cycle of synthesis, SAMA-OPfp (0.5 mmol) [Doridiphoth] J.
W. (1989), Ph.D.Thesis, Leiden, The Netherlands]
Standard protocol omitting piperidine treatment for 30 minutes in the presence of -hydroxybenzotriazole (0.5 mmol) (i.e., "Fmoc-deblocking step", which in this case would cause an unwanted S-deacetylation reaction). Wax) was used for coupling. These are called SAMA-peptides.

The positions of the peptides and their surface regions conjugated to TT are as follows: Conjugation of SAMA-peptide to tetanus toxoid was performed as follows. A solution of N-succinimidyl bromoacetate (4.7 mg, 10 μmol) in DMF (100 μl) was added to 0.1 mol sodium phosphate buffer pH 7.8 (3.5 ml).
It was mixed with a solution of tetanus toxoid (TT) (20 mg) in. After 1 hour, 1.8 ml of the reaction mixture was added with 5 mmol of EDT.
0.1 molar sodium phosphate (PE buffer) pH containing A
Sephadex PD-10 equilibrated in 6.1
Gel filtration was performed using a column [Hharmacia]. Bromoacetyl tetanus toxoid was eluted with the same buffer and collected at 3.5 ml. . A solution of bromoacetylated tetanus toxoid was added to the SAMA peptide (4.5 m
g, 3 μmol) and degassed with helium.
Then 150 μl of 0.2 mol hydroxylamine (in PE buffer, pH 6.1) was added. After 16 hours, the remaining bromoacetyl groups were blocked by the addition of 2-aminoethanethiol hydrochloride (4 μmol) in buffer, pH 6.1 (150 μl). After a further 16 hours, the peptide-TT conjugate was treated with PD-10.
Purified by column gel filtration and eluted with PE buffer, pH 6.1. Appropriate fractions were combined and stored at 4 ° C.

To determine immunological activity, 25 μg (total protein) / dose of peptide-TT conjugate was injected subcutaneously into 6-8 week old NIH outbred mice at 0 and 4 weeks. . (Note: Vaccines LBV 017-TT and LBV 018-TT
Was used at 10 μg total protein / dose). Serum was collected 6 weeks after the first dose and evaluated for antibody response in an ELISA assay [Beuver.
y) et al. (1983) Infect.immun. 40 : 369-380]. The following antigens were coated in the wells of a microtiter: outer membrane protein (OMP), purified class I OMP [Poolman, JT et al. (1989) Infection.Immun.). 57 : 1005] and unconjugated peptides. Serum bacterial activity (BC) was also measured [[Poolman, JT et al. (198
5) supra].

  The results are shown in Table 2 below.

As these data suggest, N. meni
ngitidis) class 1 and 2 OMP tested constant surface loops, loop 5 is N. mening
Itidis) appears to represent at least one region that produces antibodies that cross-react with classes 1 and 2 of many strains.

Example 7: Construction of Recombinant Flagellin Expressing Neisseria meningitidis Epitopes To generate a hybrid flagellin containing a class I Neisseria meningitidis epitope, a series of oligonucleotides and primary protein sequence data and Displayed based on data of epitope mapping. Adventitia
Two oligonucleotides based on VR1 and VR2 of P1.7.16 allow them to be cloned in single or multiple copies into the cloning region within the gene for Salmonella muenchen flagellin. Designed to. A translation stop signal was included on the non-coding strand of the oligonucleotide to facilitate screening by expressing the cloned insert.

Flagellin H1-d of Salmonella meuchen (accession number entrusted to ATCC
Plasmid vector pPX1650 containing the entire coding region and promoter region of the structural gene for
Was modified to contain several unique cloning sites suitable for insertion of oligonucleotides or gene fragments in each of the three reading frames of the flagellin gene. First, pPX1650 was digested with EcoR V, EcoR V cleaved pPX1650 twice, separated 48 base pairs, and recombined to produce plasmid pPX1651, which has a unique EcoR V cloning site, And a 16 amino acid deletion occurs in the flagellin protein. pPX1651 was identified by screening recombinants of E. coli on Western blots probed with a polyclonal antibody directed against H1-d flagellin. pPX1651 has a smaller flagellin than wild-type flagellin (of 1650),
It was identified among several candidates and evaluated by sequencing. Second, pPX1651 was restricted with BamHI and the overlapping ends were filled out with Klenow enzyme to obtain BamHI in the polylinker region of the vector.
After removing the restriction enzyme, it was recombined. As a final step, the resulting vector was digested with EcoR V and the following oligonucleotide linker was inserted: The candidates were screened for newly created BamH I sites, and some candidates with BamH I sites were
Screened for the orientation of the linker by the double-stranded DNA sequencing method. One candidate with the linker facing up was retained as pPX1647: Plasmid pPX1647 (Figure 7) was digested with BamHI and either oligonucleotide for VR1 or VR2 was cloned into E. coli cells. Screening for the desired recombinants involves digestion of the plasmid minilysate DNA with the appropriate diagnostic restriction enzyme and SDS.
-For reduced mobility on PAGE, specific flagella antiserum (H1
-D) by probing hybrid flagella and screening for expression. A certain number of resulting clones showed reduced mobility on SDS-PAGE, indicating one or more appropriate insertions of the oligonucleotide for VR1 or VR2. DN some of each
Retained for analysis by sequencing A. Clone CB1
-2 results from the tandem insertion of two copies of the VR1 oligonucleotide, and clone CB1-4 results from the insertion of four oligonucleotides. Similarly, CB2 P is V
Contains a single insert of the R2 oligonucleotide, and CB2 W shows the insert of the expected trimer, C
The B2P clone contained a single base pair change, which resulted in a Leu to Phe change in the expressed VR2 fusion protein and was not retained for further studies.
Recombinant flagellin clones in E. coli are known to react with either VR1 or VR2 epitopes [Abdillah
i) and Poolman, microvial
Pathogenesis (Micro.pathogenesis) 4: 27-32, 1988; RI
VM, Netherlands] Probing Monoclonal Ad
am-1 (P1.7) and Mn14-C11-6 (P1.7) react with hybrid flagellin containing 2 or 4 tandem inserts of VR1 but not with clones containing VR2. The weaker reaction of both monoclonals with CB1-2 than CB1-4 appears to be due to the density of the epitopes. Similarly, monoclonals 62 (P1, 16) and Mn14-c11-G (P1.16) react with the CB2 clone but not the VR1 insert. The CB2 P clone is
It does not react with any VR2 antibody, probably due to the conversion of Leu to Phe.

Each of these clones was transformed into the aroA S. dublin strain (SL5927), which has a Tn10 insertion at the H1-d position, to test hybrid flagella function. Each of the four clones gave rise to motile bacteria; the motility of the transformants was inhibited by the corresponding monoclonal antibodies, including clone CB2P, which increased the affinity of the VR2 monoclonal for epitopes in intact flagella. Indicated.
As the results show, the epitope is exposed on the surface of the cell and the antibody is accessible to the epitope.

Hybrid flagellins containing both VR1 and VR2 epitopes were created by cleaving CB1-4 or CB2W with BamHI and cloning the heterologous epitope. Clone CB12-7 and CB12-10
Results from the in-frame insertion of a single copy of the VR2 oligonucleotide behind the tandem insert of 2 or 4 VR1s respectively; clone CB21-F shows 3 of VR2.
It results from the insertion of one copy of the VR1 epitope behind the tandem copy. CB12-7 and CB12-10 are recognized only by the VR1 monoclonal antibody, and CB21-F
Only recognized by VR2 monoclonal. These results, together with analysis of the DNA revealing the predicted sequence, indicate that the density of epitopes is too low in the combined hybrids. To create a hybrid flagellin with an increased density of both VR1 and VR2 epitopes, CB12-10 was digested with BamHI and an oligonucleotide encoding VR2 was inserted. Clone 12-10-6 contains two or more tandem inserts of the VR2 epitope, resulting in a hybrid flagellin molecule in which there are four tandem copies of VR1 followed by three copies of VR2. As shown in Figures 3a and b, three hybrid flagellin vaccine candidates have the expected molecular properties. Flagellin containing four copies of VR1 (pCB1 × 4) reacts with anti-H1-d (anti-flagellin) and anti-VR1 monoclonal antibodies but not with anti-VR2 monoclonal antibody; VR2
Of flagellin (pCB2-
W) reacts with anti-H1-d and anti-VR2 antibodies but not with anti-VR1; a combination other hybrid containing a copy of VR1 and 3 copies of VR2 has anti-VR1 and anti-VR2 of both.
React with the monoclonal antibody of. The combined hybrids identified motility when introduced into the non-motile receptor S. dublin strain.

As a subunit vaccine, the goal is to obtain suitable initial vaccine candidates in high quantity and high purity. Suitable vaccine candidates include salmonella responsive to monoclonal antibodies and non-motile.
a) Can be selected from the above types of constructs based on the function of the host strain. The subunit flagellin vaccine need not retain all functional aspects of the parent flagellin, but should at least retain surface localization for purification purposes. Several subunit flagellin meningococcal vaccines were selected from the aforementioned hybrid molecules based on their reactivity to monoclonal antibodies, implying surface localization based on restoration of bacterial motility . The three flagellin vaccine candidates contained four tandem inserts of VR1, three tandem inserts of VR2, or four VR1 inserts followed by three VR2 inserts.
Since flagellin is the major protein of Salmonella, sufficient material for vaccination using established techniques for flagellin can be easily purified [Logan et al., Journal of・ Bacteriology 16
9: 5072-5077, 1987].

Example 8: Initial Purification of Recombinant Flagellin Molecules Three hybrid flagellin vaccine candidates and wild type (derived from pPX1650) were loaded into a 4 liter baffled Fernbach flask containing 1 liter LB broth. Inoculated into. 22. Bacterial culture at 37 ° C with shaking (200 rpm)
Incubated for ~ 24 hours. Under these culture conditions, most of the flagella were shed from the bacterial cell surface and localized in the supernatant medium. Flagella were separated from 6-8 liters of medium to obtain surface material. To obtain a purified flagellin preparation for vaccination, flagella filaments were harvested from the bacterial culture supernatant by the following procedure: ammonium sulphate was added to the culture supernatant to give a final solution of 50%. The solution was stirred gently at 4 ° C. for several hours and the precipitated material was stirred at 5000 rpm in a GSA rotor.
Harvested by centrifuging for 30 minutes at. The collected ammonium sulfate precipitated material was reconstituted in PBS and P
It was dialyzed for 12-15 hours at 4 ° C. against BS. The dialyzed material was subjected to high speed centrifugation in a SW-27 rotor at 100,000 xg for 1 hour to pellet the flagella filaments. The precipitated material consisted mainly of flagellin and was further precipitated by the following method.

Example 9: HPLC Purification of Recombinant Flagellin To prepare highly purified flagellin, a Salmonella genus (S) expressing a construct, particularly pCB12-10-6, was prepared.
almonella) were grown as described above and cells were
Precipitated at 0,000 G. The culture supernatant was then precipitated with 50% ammonium sulphate, centrifuged at 10,000 G and resuspended in 30 ml PBS. The resuspended precipitate was mixed with 6 mol urea, 1 mmol PMSF, 2 mmol NE.
It was dialyzed overnight at 4 ° C. against 10 mM Tris buffer (pH = 8.0) containing M, and 5 mM EDTA. The dialyzed material is then treated with two sepharoses (Se).
pharose) mini column (volume of 3.0 ml each, eluent of 4.0 ml). Column contains 6 moles of urea
50 mM NaCl in 10 mM Tris (pH = 8.0)
, Then eluted with 1 mol NaCl in 10 mmol Tris (pH = 8.0) containing 6 mol urea (5x). 50
The first 4 eluate collections (20 ml) of mmol NaCl were pooled and dialyzed at room temperature against 1.0 ml of acetate buffer (pH = 4.0) in 6 molar urea. The dialyzed fraction was then applied on a TSK SP PW cation exchange HPLC column (75 mm x 300 mm). The column was eluted with a mobile phase consisting of 10 mmol acetate (pH = 4.0) containing 6 mol urea. Gradient 0-300 mmol NaCl, 10 mmol acetate containing 6 mol urea (pH =
4.0) at intervals of 5-30 minutes. After 30 minutes, the gradient went from 300 mmol in 10 mmol acetate containing 6 mol urea to 1 mol NaCl over the next 5 minutes. Flagellin constructs are collected in approximately 24 minutes,
This corresponds to about 200 mmol NaCl. Fractions were dialyzed against PBS and the purity determined for the substance was established by Western blot using anti-flagellin antibody. Representative HPLC analysis and SDS-PAGE are shown in Figures 8 and 9, respectively.

Example 10: Preparation of Neisseria meningitidis-flagellin glycoconjugate Group C meningococcal capsular polysaccharide (GCM CPS: lot # 86
NM01) is essentially Bundle et al., Journal of Biological Chemistry (J.Biol.Che).
m.), 249 : 4797-801, 1974.

Neisseria meningitidis strain C11 is a product of the Walter Reed Army Institute (Walter
Reed Army Institute) (Washint DC). The strain was pre-cultured twice on sheep blood agar plates and then liquid seed medium Neisseria chemically defined medium, NCDM [Kenney et al.
Bull.WHO 37 : 469-73, 1967]. Finally, 40 1 of liquid medium in the fermentor was inoculated with the liquid preculture. The purity of the strain was checked at each stage. After centrifugation, the supernatant was precipitated by adding Cetavlon to a final concentration of 0.1%, and the insoluble complex redissolved in cold 1 molar calcium chloride (CaCl ₂ ) [Gottschrich ( Gotschlich) et al., Journal of Experimental Medicine (J.Ex)
p.Med.) 129 : 1349-65, 1969]. Ethanol (96%)
Added to a final concentration of 25% (v / v). After 1 hour, the suspension was collected and its concentration was increased to 80% (v / v). 1
After hours, centrifugation (20 min, 5,000 g) gave a precipitate, which was washed successively with absolute ethanol, acetone, and diethyl ether, then in the vacuum desiccator, the presence of phosphorus pentoxide (P ₂ O ₅ ). Dried down to constant weight. The crude CPS was stored at -20 ° C.

CPS was then dissolved in a buffer of sodium acetate (1.10 dilution of saturated solution, pH 7.0) and extracted 4 times with hot phenol to obtain a pure preparation [Westphal et al. Z.Naturforsch. 7b: 148-55, 195
2]. After dialysis of the combined aqueous phase against 0.1 M CaCl ₂ and subsequent centrifugation (3-5 hours, 100,000 g), a final ethanol precipitation was carried out on the clear supernatant and the resulting precipitate as described above. Washed with an organic solvent,
And dried. The pure CPS was then stored at -20 ° C.

After each step of purification, the CPS was converted to the carbohydrate N-acetylneuraminic acid, NANA [Svennerhold,
Bio Himica Et Bio Physica Actor
m.Biophys.Acta), 24 : 604, 957], O-acetyl (Hesrtin, Journal of Biological Chemistry (J.Biol.Chem.), 180 : 249, 194.
9], and analyzed for protein (260 nm) content,
The molecular weight was then checked by gel filtration.

Group C meningococcal capsular polysaccharide (GCM CPS) was simultaneously depolymerized and activated via oxidation of sodium periodate (NaIO ₄ ) in aqueous buffer [Anderson (Anderson
erson) et al., Journal of Immunology (J.Immun
ol), 137 : 1181-6, 1986; Eby et al., Pediat.R.
es.20: 308A, 1986; Anderson et al., J. Ped.
iatr.111 (5): 644-50, 1987; Anderson (Anderso
n), U.S. Pat. No. 4,762,713; 1988]. The reaction was monitored by high performance gel permeation chromatography (HPGPC) in aqueous eluent using detection of ultraviolet (UV) and refractive index (RI). The reaction was stopped and the activated oligosaccharide (GCM OS) was desalted by low pressure gel permeation (GPC) in water, then lyophilized. The solution was then prepared in water and subsequently frozen for temporary storage. GCM
The OS and flagellin pCB12-10-6 mixed in a buffer of neutral aqueous, and the bonding is initiated by the addition of sodium cyanoborohydride (NaBH ₃ CN) [Anderson (Anderson), U.S. Pat. Nos. 4,762,713; 1988; U.S. Pat.No. 4,673,547, 1987; U.S. Pat.No. 4,761,283, 198
8]. The reaction was carried out for 5 days, during which time it was monitored by HPGPC. It was finally stopped by a dialysis / centrifugation microconcentrator. The final preparation was stored cold in the presence of thimerosal to prevent bacterial growth. The resulting glycoconjugate not only provides a mechanism to present the expressed VR1 and VR2 meningococcal epitopes to the immune system, but also serves as a carrier molecule for the presentation of meningococcal oligosaccharides. .

The following conditions were used in the preparation of the conjugate. Purified flagellin pCB12-10-6 was dissolved in 15% sucrose (3.5 mg / ml) and then stored at -20 ° C. GCM CPS (9.7 mg; final concentration: 5 mg / ml) was stirred in the dark with 0.50 molar sodium phosphate buffer (pH 6.
2-6.5) at room temperature with 100 mmol of NaIO ₄ . Aliquots (100 μl) are removed at regular intervals, the reaction is stopped by the addition of ethylene glycol (10 μl), and the analysis is carried out in 0.2 mol phosphate-physiological saline buffer (PBS; 0.2 mol sodium phosphate, 0.9%).
Waters (Milford, MA) Ultrahydrogel (Ultrah) in NaCl, pH 7.8)
ydrozel ^R ) 250 + 120 (2 coupled columns; 2x
300 mm x 7.8 mm) HPGPC at a flow rate of 0.8 ml / min using UV (206 nm) and RI detection. After 2.5 hours, the reaction was stopped by addition of ethylene glycol (1/10 of the reaction volume), and GCM OS of water Bio - Rad (Bio-Rad) (Richmond, CA) Bio - Gel (Bio-Gel ^R ) P-2 (200-400 mesh, 3
(0 cm x 1.5 cm) at about 18 ml / hour. Fractions were collected (1.2 m) and NANA carbohydrate N-acetylneuraminic acid (NANA) [Barry et al., Journal of.
General Microbiology (J.Gen.Microbi
l.) 29: 335-52, 1962] and the aldehyde [Porr (Porr
o) et al., Analytical Biochemistry (Analyt.
Biochem.) 118: 301-306, 1981]. Positive fractions were pooled and lyophilized. Desalted GMC OS (4.7 mg) was then dissolved in water (10 mg / ml) and frozen at -20 ° C.

Solutions of GCM OS and pCB12-10-6 were analyzed by HPGPC (UV at 206 and 289 nm, respectively) before freezing and before conjugation. No digestion occurred during storage, corroborated by the exact similarity of the elution profiles.

GCM OS (2 mg; final concentration: 2.6 mg / ml) and flagellin pCB12-10-6 (2.3 mg; final concentration: 3 mg / ml) were added to a polypropylene tube in 0.4 molar sodium phosphate buffer (pH 7.
0), and NaBH ₃ CN was added (12 μmol) to initiate conjugation [Anderson, US Pat. No. 4,762,713; 1988; US Pat. No. 4,673,547; US Pat. No. 4,761,283]. The reaction mixture was at room temperature for 1 day,
It was then left at 35 ° C. for 4 days without stirring. The reaction was monitored at different stages of HPGPC (ultraviolet at 280 nm) and finally stopped in a microconcentrator by dialysis / concentration. The final concentration is NANA [Barry (Barr
y) et al., Journal of General Microbiology 29: 335-52, 1962] and (0.09 mg at 0.12 mg / ml) and protein [Lowry et al., Journal. Of Biological Chemistry (J. Biol. Chem.) 193: 265-275, 195.
1] (1.12 mg; 1.45 mg / ml) content was analyzed. It was then stored at 4 ° C. in the presence of thimerosal (0.01%, w / v) to prevent bacterial growth.

The zygote was also prepared by SDS-PAGE (staining of silver nitrate).
And Western blot analysis. Some high molecular weight bands are pure pCB12-10-6 on the gel.
Appeared near the stacked wells above the band
The latter was evidence that cross linkage occurred during conjugation. On Western blot analysis, each band was shown to be reactive with the antisera used (anti-GCM-VR1 and VR2), demonstrating covalent conjugation.

Example 11: Conjugation of N. meningitidis peptides to CRM and bovine serum albumin Peptides designated M20 and M21 were produced using tBoc chemistry by solid phase synthesis on an ABI type peptide synthesizer,
Bifunctional cross-linking agent, sulfosuccinimidyl (4-iodoacetyl) aminobenzoate [Sulfo SIA
B; Purchased from Pierce] [Weltman JK et al. (1983) Bio Technigues 1, 148-15.
According to the modified method of [2], CRM ₁₉₇ [Anderso (Anderso
n), U.S. Pat. No. 4,762,713]. Briefly, CRM ₁₉₇ was activated by sulfo SIAB,
It produced an amide bond between the amino group of SIAB and CRM _197.
Continuous spacer with carboxy-terminal cysteine residue after removal of unreacted cross-linker from activated CRM ₁₉₇ by gel filtration (underlined letters)
Containing peptides (M20 or M21) were mixed with activated CRM and incubated at room temperature for 2-4 hours. After the reaction, the conjugated material was extensively dialyzed against PBS at 4 ° C.

The sequence of the M20 peptide (VR2 epitope) is as follows: The sequence of the M21 peptide (VR1 epitope) is as follows: The conjugated material was subjected to SDS-PAGE and transferred to PVDF membrane [Immobilon, Millipore],
It then reacted with specific monoclonal antibodies that recognize VR1 and VR2 epitopes. Figures 10a and 10b show
VR1 and VR2 specific monoclonal antibodies (Adam I, G2
-D12-8 (P1.7), MN5-C11-G (P1.16) and MN14
-C11-6 (P1.7)] pool against M20 and M21
3 shows a Western blot analysis of CRM ₁₉₇ conjugates of.

To assay the response of antibodies to the M20 and M21 peptides by an enzyme-linked immunoassay procedure, BSA conjugates were prepared using Bernatowicz and Matsueda [Analyt. Biochem. 155, 95-102. (1986)] by using a different bifunctional crosslinker, N-succinimidyl bromoacetate. Covalent coupling of peptides to proteins was confirmed by Western blotting of electrophoresed samples as described for _CRM197 conjugates.

Example 12: M20 and M21-CRM ₁₉₇ junction epitopes VR1 and VR2 to the holding CRM ₁₉₇ of the activity of T cells by conjugate CRM ₁₉₇
Bixler and Atassi to determine whether they adversely affect T cell recognition
Assays for T cell proliferation were performed as previously described in [Immunol. Commun. 12: 593, 1983]. Briefly, 50 μg of native CRM1 emulsified in SJL / j mice in CFA.
Immunized with 97. After 7 days, lymph nodes were removed, cultured in RPMI, and various concentrations of protein (0.05-100.
0 μg / ml) and peptides. After incubation for 3 days, the cultures were pulsed with [ ³ H] -thymidine for 16 hours and then harvested for counting.

As shown in Table 3, a comparison of _CRM197 and CRM197- _{pseudozygotes} shows that the conjugation procedure itself does not alter T cell recognition of the protein. M20 and M21-CRM ₁₉₇
The zygote-induced T cell response is essentially equal to or greater than the response elicited by CRM ₁₉₇ itself, and recognition of T cell epitopes on CRM ₁₉₇ is adversely affected by peptide conjugation. It is shown that it is not done. Responses of control substances to ConA, LPS and tetanus toxoid were as expected.

Example 13: Immunogenicity of the conjugate and recombinant meningococcal b vaccine Recombinant flagellins expressing the Neisseria meningitidis VR1 and / or VR2 epitopes are described in Examples 7, 8 and 9. , Prepared and purified. In addition, synthetic peptides representing the meningococcal epitopes VR1 and VR2 were synthesized, coupled to the carrier molecule CRM ₁₉₇ , and purified as in Example 12. The vaccine was formulated with each of these materials at a protein concentration of 10 or 100 μg / ml for each of the components. The vaccine composition also contains aluminum phosphate at 1 mg / ml,
Alternatively, unless otherwise specified, it was formulated with Freund's complete adjuvant or contained no auxiliary substances.

To assess immunogenicity, outbred Swiss Webster mice were immunized intramuscularly at week 1 and week 2 with 1 or 10 μg protein / dose. Serum was collected at 2 week intervals, pooled for assay, and outer membrane complex (OMC) by ELISA,
Purified OMP (M21-BSA), VR2 coupled to BSA
Peptide (M20-BSA), wild-type flagellin, and CR
_Screened for activity of antibodies to _M197 .
Table 4 shows the results of the ELISA performed on the sera obtained at the 6th week.

¹ All pre-bleed values at or below the 1/100 dilution assay lower limit.

All ² vaccines were formulated using 1 mg / ml aluminum phosphate unless otherwise stated.

Alternatively, various vaccines were evaluated for immunogenicity in 6-8 week old NIH outbred mice. Mice were immunized subcutaneously with vaccine at weeks 0 and 4 at 100 μg (total protein) / dose and sera collected at week 6. Serum was treated with EL as described in Example 6.
Evaluation was performed using the antigen by the ISA assay. Bactericidal activity was measured as in Example 6. The results are shown in Table 5.

Recombinant flagellins containing VR1, VR2 or both VR1 and VR2 cassettes are effective in eliciting an antibody response that is cross-reactive with purified P1.16 and to a lesser extent OMC. Met.
Sera from animals immunized with 10 μg of pCB1-4 or pCB2-w elicited antibodies that bound their respective peptide-BSA conjugates and cross-reacted with P1.16 and OMC. Similar results were obtained using constructed pCB12-10-6 containing both Neisseria meningitidis epitopes.
Moreover, each construct elicited significant anti-flagellin titers as well. In contrast, control wild-type flagellin elicited only antibodies responsive to flagellin itself. Serum collected prior to immunization showed no pre-existing response to the substances evaluated.

The data also demonstrate the benefit of formulating recombinant flagellin using alum or other adjuvants such as CFA. Construct pCB12-10-6 was formulated with or without the addition of aluminum phosphate. Table 2
As shown in, pCB12-10-6 was capable of eliciting peptide conjugates as well as purified P1.16 and antibody responses reactive with OMC alone. In contrast, the same construct was able to elicit a larger antibody response at equal doses when formulated with alum. Similarly, recombinant flagellins pCB1-4 and pCB2-w were also cloned into CFA
It was mixed with. Again, an equal or higher antibody titer is GP
Observed in the presence of A.

The results of immunogenicity studies using N. meningitidis VR1 and VR2 conjugates are also shown in Table 4. Both M20 and M21
-CRM ₁₉₇ conjugates as well as mixtures containing equal amounts of both were able to elicit anti-CRM ₁₉₇ responses as well as anti-class I OMP responses.

As these preliminary data show, epitopes of the variable region of class I OMPs, either chemically conjugated to the carrier or genetically fused to the carrier, can elicit an immune response. New epitope
Carrier conjugates are made using standard techniques to produce vaccines, eg, 1) larger epitopes,
It is possible to enhance the immune response to 2) the use of peptides with multiple epitope repeats and / or 3) the use of different carriers.

Example 14: Meningococcal human - Preparation GCM CPS serum albumin sugar conjugate depolymerized by acid hydrolysis and subsequently the resulting GCM OS activated via NaIO ₄ oxidation in an aqueous buffer . The reaction was monitored by HPGPC in aqueous eluent using detection of UV and RI. Each of the reactions was followed by GPC desalting in water. GPC OS and human albumin (HA) were mixed and conjugated essentially as described for the N. meningitidis-flagellin glycoconjugate in Example 10. The final preparation was stored in the cold in the presence of thimerosal to prevent bacterial growth.

The following experimental conditions were used in the preparation of the conjugate. Human albumin [HA; Sigma ^R , St. Louis, Missouri] was dissolved in 15% sucrose (10 mg / m
l), then stored at -20 ° C. GCM CPS (Lot # 86 NM 01; 106mg; final concentration: 10mg / ml) with 0.1N HCl
Hydrolyzed at 50 ° C. with stirring in. Aliquots (25 μl) were removed at regular intervals, the reaction was stopped by the addition of sodium hydroxide (NaOH) and analyzed by HPGPC as described. After 3 hours and 40 minutes, the reaction is N
It was stopped by the addition of aOH and GCM OS was desalted by GPC. Fractions were collected (1.2 ml) and analyzed as above. Positive fractions were pooled and lyophilized.
Desalted GCM OS (89 mg) was then stored at -20 ° C. By oxidizing GCM OS (11.8 mg; final concentration: 5 mg / ml) with 2 mmol NaIO ₄ in 0.05 mol sodium phosphate buffer (pH 6.2-6.5) at room temperature in the dark with stirring. An activated OS was prepared. After 30 minutes, the reaction was stopped by adding ethylene glycol.
Analysis of HPGPC showed no decrease in the molecular weight of OS during activation. Desalting and colorimetric analysis was then performed as described above. The resulting activated GCM OS (8.8 mg) was dissolved in water (10 mg / ml) and frozen at -20 ° C.

Both GCM OS and HA solutions were subjected to HPGPC (UV at 206 and 280 nm, respectively) before freezing and prior to conjugation.
Was analyzed by. No degradation occurred during storage, as confirmed by the exact similar elution profile.

GCM OS (6 mg; final concentration: 2.5 mg / ml) and HA (12 mg;
(Final concentration: 5 mg / ml) in a polypropylene tube in 0.4 molar sodium phosphate buffer (pH 7.0) and mixed with NaB
H ₃ CN (60 μmol) was added to initiate the bonding [Anderson, US Pat. No. 4,762,713; 1988; US Pat. No. 4,673,547, 1987; US Pat. No. 4,761,283, 198.
8]. The reaction mixture was left unstirred for 1 day at room temperature and then for 4 days at 1535 ° C. The reaction was monitored at different stages of HPGPC (ultraviolet at 28 nm) and finally stopped in a microconcentrator by dialysis / concentration. Final concentration to NANA [Barry
Et al., Journal of General Microbiology 29 : 335-51, 1962] (2.07m
g, at 0.86 mg / ml) and (at 0.12 mg / ml 0.0
9 mg) and protein [Lowry et al., Journal of Biological Chemistry (J. Biol. C.
hem.) 193 : 265-275, 1951] (at 9.51 mg; 3.96 mg / ml). It was then stored at 4 ° C. in the presence of thimerosal (0.01%, w / v) to prevent bacterial growth.

The zygote was also prepared by SDS-PAGE (staining of silver nitrate).
And Western blot analysis. Diffusion bands appeared on the gel and covered a significantly wider range of molecular weights than pure HA. On Western blot analysis, this band was shown to be reactive with the antiserum used (anti-GCM), demonstrating covalent attachment of the conjugate.

Example 15: Immunogenicity of a meningococcal oligosaccharide-recombinant flagellin vaccine A meningococcal oligosaccharide-recombinant flagellin vaccine was prepared as described above and 100 μg.
Protein / ml. In addition, a vaccine composition containing 1 mg / ml of aluminum phosphate or Freund's complete adjuvant in addition to the glycoconjugate was prepared.

To assess immunogenicity, outbred Swiss Webster mice were immunized intramuscularly with 10 μg of protein at weeks 0 and 2. Serum was collected at weeks 0 and 2 and then at weekly intervals until week 6. After collection, pooled serum samples were assayed by ELISA for activity of antibodies against meningococcal C oligosaccharide conjugates against human serum albumin, OMC, P1.16, CB1 and CB2-BSA conjugates and flagellin.

The MenC-CB12-106 glycoconjugate is expressed in recombinant flagellin and oligosaccharides and N. meningitidis B
It was effective in eliciting an immune response reactive with both OMP epitopes. As shown in Table 5B, mice immunized with 1 μg of MenC-CB12-106 in complete Freund's adjuvant for as little as 3 weeks after entering the study were tested for MenC-HSA, OMP and both CB1 and CB2. It had a detectable antibody to the epitope. Furthermore, all MenC-CB12-106 preparations elicited an antibody response to MenC-HSA that was greater than that observed after immunization with MenC-CRM197, regardless of adjuvant.

1 The titer for the initial pre-bleed (week 0) sample is <
It was 100.

Aluminum diphosphate was used as an adjuvant at 1 mg / ml.

Example 16: Class I OMP T Cell Epitopes and Their Identification An effective vaccine must contain one or more T cell epitopes. The T cell epitope within the protein has been reported by Margalit et al., Journal of Immunology (J. Immunol) 138 : 2213 (198
7) or Rothbard and Tyler (Tay
lor), EMBO Journal (J.) 7: as described in 93 (1988), it can be predicted. These prediction methods are based on the N. meningitidis strains P1.7, 16, P1.6.
And the amino acid sequence of class I OMP of P1.5. Segments of sequences containing potential T cell epitopes identified by these methods are shown in Tables 6 and 7. The predicted peptide was synthesized by standard FMOC procedures, purified by standard methods and identified as shown in Table 8.

To determine whether the predicted peptides actually contained T cell epitopes, their ability to stimulate human peripheral blood lymphocytes (PBLs) was tested by lymphocyte proliferation assay. Simply put, peripheral blood
From HLA type normal volunteers or MPC-2
[Poolman, JT et al., Antonie van Leeuwenhoek 5
3 : 413-419, 1987], P1.16, 15, Class 4 OMP
And containing group C polysaccharides were collected from previously immunized volunteers. Lymphocytes from peripheral blood Ficoll-Hypaque [Pharmacia
Fine Chemicals
AB, Uppsala, Sweden] and 10%
Of heat-inactivated and pooled human AB serum supplemented RPMI 1640 [Gibco Laboratories (Gibco Laboratories
atories), Paisley, Scotland] 1 x 10 ⁵
Cultured in cells / well. Cultures were challenged with various concentrations of the predicted T cell epitope (0.05-10 μg / ml). After in vitro challenge, cultures were incubated for 6 days and then pulsing (18 hours) with 0.5 μCi [ ³ H] -thymidine. Cultures were then harvested and counted by liquid scintillation. Data are expressed as stimulation index, where the index was calculated as the ratio of CPM obtained in the presence of antigen to CPM obtained in the absence of antigen.

As shown in Table 9, 10 out of 16 predicted peptides
Showed some ability to stimulate T cells. These are 16
-34, 47-59, 78-90, 103-121, 124-137, 151-15
The peptides identified in 8, 176-185, 223-245, 276-291 and 304-322 are included. In some cases, the peptides stimulated a response in both immunized as well as non-immunized individuals. Responses in non-immunized individuals can be attributed to previous asymptomatic infections.

In the case of T cell epitopes identified as regions 176-185, enhanced T cell responses were observed after addition of monoclonal antibody MN5C11G (P1.16). Briefly, PBLs were challenged in vitro with a synthetic peptide containing the region 175-185 or with this peptide mixed with varying dilutions of MN5C11G. As shown in Table 10,
An enhanced T cell response was observed after addition of MN5C11G, showing that the monoclonal antibody recognized region 176-185 and facilitates the presentation of the peptide to the immune system. Thus, the T and B cell epitopes are
It was established that they were matched or found on flanking sequences within the class I OMP.

In some cases T cell lines and clones were established from individuals who responded to various peptides. Briefly, the T cell line was obtained by culturing isolated lymphocytes at 1 × 10 ⁶ cells / ml in 24-well plates. RPMI-1640 medium supplemented with 10% human serum also contained 12 U / ml recombinant IL-2 [Boehringer]. In addition, 5 × 10 ⁴ homologous irradiated (3,000R) antigen-representing cells (APC) were also added to each well. In some cases, APC is HLA
Obtained from a compatible donor. From the lineage, T cell clones were isolated by limiting dilution at a frequency of 0.5 cells / well. Clones were maintained by stimulation with antigen for 2 weeks in the presence of irradiated APC and IL-2 (2U / ml). Clones were tested essentially by the assay for lymphocyte proliferation as described above, except that the clones were 1x1 in the presence of irradiated APC.
0 were cultured in ⁴ cells / well.

The clones obtained as described above, in vitro,
The OMPs from 7 different Neisseria meningitidis strains were challenged. Table 11
The clones recognized T cell epitopes or a common epitope for the seven OMPs examined, as shown in FIG. The responsiveness of these clones to various peptides has not yet been determined, but nevertheless shows commonality of T cell epitopes among various strains. Now that these clones have been established and identified,
The reactivity of those peptides will represent T cell epitopes for various uses.

Responses have the following scores:-, SI <2; ±, 2 <
SI <and +, SI> 3.

The underlined region shows the sequence recognized by the monoclonal antibody MN5C11G.

Example 17: Construction of a protein model for the membrane topology of class I OMPs and comparison to other pathogenic Gram-negative porin proteins for the development of vaccines Several structures of E. coli outer membrane proteins were recognized. The model was constructed using the principle [Vogel, H. et al. (1986) supra; Ferenc
i), T. et al. (1988) supra; and Tommasse (Tommasse).
n), J. (1988) supra]. The central assumption is that the protein segments that span the other membranes form beta-sheets. Specifically, in the case of class 1 proteins, the division in exposed transmembrane segments was reached in the following way: 1. Amino acids of class 1 proteins (subtype P1.16) and N. gonorrhoeae PIA and PIB proteins Comparison with the amino acid sequence of [Carbonetti, NH et al. (1987)
PNAS 84 : 9094; Carbonetti, NH et al. (19
88) PNAS 85 : 6841; and Gottschlic (Gotschlic
h) EC et al. (1987) PNAS 84 : 8135] reveal 34% identity. In this model, the variable sequences form the exposed parts of the surface, whereas the conserved regions are located mostly in the adventitia and periplasm. Thus
The latter two regions make up 58% of the residues conserved among all proteins.

2. The maximum hydrophilicity corresponds to the areas exposed in the hydropathic profile [Kyte, J. et al. (1982) Journal of Molecular Biology 157 : 105]. It was observed.

3. Transmembrane segments are able to preferentially form bipolar beta-strands of 9-12 bases, with at least one side consisting entirely of hydrophobic residues. These conditions are met in 12 of the 16 membrane-spanning segments.

4. The number of residues on the periplasmic side is minimal.

Figure 11 shows a model for folding of class 1 proteins in the outer membrane. The sequence shown is for subtype P1.16. The upper part of the figure shows the exposed area of the surface, whereas the central part shows the hypothesized transmembrane segment, the length of which is set to 10. Amino acids have been shown to alternate, where they are capable of forming bipolar beta-strands. This model contains eight surface loops, where the first and fourth loops contain type-specific and protective variable region epitopes. These epitopes are capable of eliciting a protective immune response, as shown when formulated into a vaccine. Loop 5
Is constant and has been shown to elicit cross-reactive antibodies to other OMPs and is useful in the formulation of vaccines.

The variable epitope regions of one or two of the individual proteins are located on the so-called surface loops of these membrane proteins. Such porin open coat proteins contain more than two surface loops. As this means,
Surface loops with nearly identical amino acid sequences are similarly abundant in different class 1 outer membrane proteins. This paves the way for the common use of common peptides of class 1 outer membrane proteins for vaccine purposes as well. More specifically, a schematic two-dimensional model of the Neisseria meningitidis class 1 outer membrane protein P1.16 is illustrated in FIG. This model contains eight surface loops, where the first and the fourth generation loops contain type-specific epitopes, as shown based on the results of strain subdipes. First
The surface loop of represents the above-mentioned certain region. Counteracting a certain region of loop 5 is N. meningitidis
It appears to react with the OMP complex. The amino acid sequence of class 1 OMP, which has just been induced, is shown in N.men.
ingitidis) class OMP [Murakami, K. et al., (1989)
Infection and Immunity (Infect.Imm
un.) 57 : 2318] and Neisseria gonorrhaeae
Porin PIA and PIB proteins. Class I
On the same principle used for the OMP's model, the sequences were aligned as follows: Structural similarities are shown in the regions of transmembrane and surface loops. Using the information available here for class I OMPs and information based on loop size, position, and inter-species amino acid homology or heterogeneity in the loop region of a particular porin protein, N. gonorrhaea
Prediction of epitopes to be incorporated into vaccines for other pathogenic Gram-negative bacteria, including e) is possible. These epitopes can be evaluated for vaccine purposes using the same methods used for Class I OMPs.

Entrustment of microorganisms N. meningitidis strain H4476 (B: 15; P1.7, 1)
6) The Central Bureau Bureau on December 11, 1989
Bour Simmel Kulturen (Centraal Bureau voor
Schimmelculturen (CBS), commissioned in Baarn, The Netherlands, and has accession number CBS 635.89. N. meningitidis class 2/3 OMP deficiency mutations
HIII-5 was deposited in CBS, Baarn, the Netherlands on December 11, 1989 and has accession number CBS 636.89.

Equivalent Embodiments A person of ordinary skill in the art would recognize or be able to ascertain using no more than routine experimentation, many embodiments that are equivalent to the particular embodiments of the invention described herein. . Such equivalent embodiments are intended to be covered by the following claims.

Front page continuation (51) Int.Cl. ⁷ Identification code FI C12P 21/02 C12R 1:36 // (C12N 1/21 1:42 C12R 1:36) C12N 15/00 ZNAA (C12N 1/21 C12R 1 : 42) (C12N 15/09 ZNA C12R 1:36) (31) Priority claim number 89.01612 (32) Priority date June 26, 1991 (June 26, 1989) (33) Country claiming priority Netherlands (NL) Microbial Accession No. CBS 635.89 Preliminary Examination (73) Patent Holder 502272309 De Start del Nederlanden Würtegen Wooldaisige de minister Vuan Weerzin, Volksghe Sondheide en Kurturd 3720 Beerbirt Hoven Peer Boxus 1 Antony van Leeuwen Hokran 9 (72) Inventor Cayde, Robert Sea, Giunia California, California 9412 1 San Francisco Twenty-Fifth Avenue 590 (72) Inventor Paradeiso, Peter Earl, New York, USA 14534 Pittsuf Aude Gilf Audeway 6 (72) Inventor Paul Mann, Jan Tei Netherlands 1151 AKEBREQUEIN WATERLAND RATEINDE 8 (72 ) Inventor Hogelhout, Peter The Netherlands 2805 Eszedgouda Idenburgstraat 13 (72) Inventor Werz, Emanuel Jeiay Eich Jei The Netherlands 3583 Ehdavryu Utrehit Mauritstraat 106 (72) Inventor Van der Rei, Peter 3583 Aem Utrecht Adriaan The Netherlands Holland Osterderan 124 (72) Inventor Hetzkels, Jyon Edward Hampshire Esau 51 6 Gitei Romsey Wee Touero Woo Alan way 6 (72) inventor Clark, Ian Nicholas UK Hanpushiya-Southampton N'esuo 1 8 Deiaru-Rounamu's Sulfur Nii Hurst Abeniyu 15 (56) Reference INFECTION AND IMM UNITY (1987) Vol. 55, No. 11, p. 2734-2740 (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 15/00-15/90 A61K 39/095 A61P 31/04 A07K 14/22 C12N 1/21 C12P 21/00-21 / 08

Claims (28)

(57) [Claims] 1. An outer membrane vesicle isolated from a recombinant or mutant Neisseria meningitidis strain that is negative for class 2/3 outer membrane proteins, said vesicle comprising at least one marrow. A meningococcal class I outer membrane protein, wherein the meningococcal class I outer membrane protein comprises A vaccine effective against meningococcal disease, which comprises any one of the amino acid sequences represented by: 2. A substantially pure fragment of a Neisseria meningitidis class I outer membrane protein, said protein consisting of any one of the amino acid sequences of claim 1 and comprising CNBr. A fragment produced by cleavage and having a molecular weight of about 25 kD. 3. A substantially pure fragment of a Neisseria meningitidis class I outer membrane protein, wherein the protein consists of one of the amino acid sequences according to claim 1. Manufactured by and about
A fragment characterized by having a molecular weight of 17 kD. 4. YYTKNTNNNL, NFVQQTPQSQP, YYTKDTNNNL, YTKD
TNNNLT, TKDTNNNLTL, KDTNNNLTLV, AQAANGGASG, QAANGGASG
Q, AANGGASGQV, ANGGASGQVK, NGGASGQVKV, GGASGQVKVT, GASG
QVKVTK, ASGQVKVTKV, SGQVKVTKVT, PAHYTRQNNT, AHYTRQNNT
D, HYTRQNNTDVF, TRQNNTDVFV, RQNNTDVFVP, QNNTDVFVP, QNNT
DVFVPA, NNTDVFVPAV, ANVGRNAFELFLIGSATSDEAKG, NIQAQLTE
QPQVTNGVQGN, TKISDFGSFIGFK, VSVAGGGASQWGN, TLRAGRVANQ
FDDASQAIN, DSNNDVASQLGIFK, GGFSGFSG, LSENGDKAKTKNSTTE
And a substantially pure oligopeptide having an amino acid sequence selected from the group consisting of VPRISYAHGFDLIERGKKG. 5. A substantially pure meningococcal class I outer membrane protein consisting of one of the amino acid sequences according to claim 1. 6. A peptide comprising an anti-meningococcal outer membrane protein antibody bound to a carrier polypeptide, said peptide comprising YYTKNTNNNL, HFVQQTPQSQP, YYTKDTN.
NNL, YTKDTNNNLT, TKDTNNNLTL, KDTNNNLTLV, AQAANGGASG, QA
ANGGASGQ, AANGGASGQV, ANGGASGQVK, NGGASGQVKV, GGASGQVK
VT, GASGQVKVTK, ASGQVKVTKV, SGQVKVTKVT, PAHYTRQNNT, AHY
TRQNNTD, HYTRQNNTDVF, TRQNNTDVFV, RQNNTDVFVP, QNNTDVFV
P, QNNTDVFVPA, NNTDVFVPAV, ANVGRNAFELFLIGSATSDEAKG, NI
QAQLTEQPQVTNGVQGN, TKISDFGSFIGFK, VSVAGGGASQWGN, TQRA
GRVANQFDDASQAIN, DSNNDVASQLGIFK, GGFSGFSG, LSENGDKAKT
A gene fusion peptide or protein selected from the group consisting of KNSTTE and VPRISYAHGFDLIERGKKG. 7. A meningococcal class I of more than one subtype that is negative for class 2/3 outer membrane proteins.
An outer membrane vesicle isolated from a recombinant or mutant meningococcal strain expressing an outer membrane protein, wherein the vesicle is 1
More than two subtypes of meningococcal class I outer membrane proteins, wherein the meningococcal class I outer membrane protein consists of any one of the amino acid sequences of claim 1. An effective vaccine against meningococcal disease. 8. A) a substantially pure meningococcal class I outer membrane protein consisting of any one of the amino acid sequences of claim 1, b) CNBr, endoLys-C, endoAgr-C or Staphylococcus VR-protease was used to produce meningococcal class I
A fragment of the meningococcal class I outer membrane protein of claim 1 produced by cleaving the outer membrane protein, and c) an amino acid sequence selected from the group consisting of YYTKNTNNNL, YYTKDTNNNL and HYTRQNNTDVF. A vaccine against meningitis disease, which comprises a component selected from the group consisting of peptides. 9. A class I outer membrane protein, fragment or peptide is complexed with a meningococcal polysaccharide selected from the group consisting of A, B, W-135 and Y polysaccharides (co
njugate) The vaccine according to claim 8. 10. The vaccine according to claim 8 or 9, wherein the class I outer membrane protein, fragment or peptide is conjugated to a carrier molecule comprising a T cell epitope of the carrier protein. 11. A vaccine according to any of claims 7 to 10 for inducing a protective immune response in a pharmaceutically acceptable vehicle. 12. A meningococcal class I outer membrane protein consisting of an antigenic carrier polypeptide, and a) any one of the amino acid sequences of claim 1, b) CNBr, endoLys-C, endoArg. -C or Staphylococcus V8-protease with meningococcal class I
A fragment of the meningococcal class I outer membrane protein of claim 1 produced by cleaving the outer membrane protein, and c) YYTKNTNNNL, HFVQQTPQSQP, YYTKDTNNNL, YTKDTNNNLT, T.
KDTNNNLTL, KDTNNNLTLV, AQAANGGASG, QAANGGASGQ, AANGGAS
GQV, ANGGASGQVK, NGGASGQVKV, GGASGQVKVT, GASGQVKVTK, AS
GQVKVTKV, SGQVKVTKVT, PAHYTRQNNT, AHYTRQNNTD, HYTRQNNT
DVF, TRQNNTDVFV, RQNNTDVFVP, QNNTDVFVP, QNNTDVFVPA, NNT
DVFVPAV, ANVGRNAFELFLIGSATSDEAKG, NIQAQLTEQPQVTNGVQG
N, TKISDFGSFIGFK, VSVAGGGASQWGN, TLRAGRVANQFDDASQAIN,
DSNNDVASQLGIFK, GGFSGFSG, LSENGDKAKTKNSTTE and VPRI
An antigenic complex, comprising a polypeptide selected from the group consisting of peptides containing an amino acid sequence selected from the group consisting of SYAHGFDLIERGKKG. 13. A recombinant microorganism capable of expressing more than one class I outer membrane protein of Neisseria meningitidis consisting of any one of the amino acid sequences of claim 1. 14. YYTKNTNNNL, HFVQQTPQSQP, YYTKDTNNNL, YT
KDTNNNLT, TKDTNNNLTL, KDTNNNLTLV, AQAANGGASG, QAANGGAS
GQ, AANGGASGQV, ANGGASGQVK, NGGASGQVKV, GGASGQVKVT, GAS
GQVKVTK, ASGQVKVTKV, SGQVKVTKVT, PAHYTRQNNT, AHYTRQNNT
D, HYTRQNNTDVF, TRQNNTDVFV, RQNNTDVFVP, QNNTDVFVP, QNNT
DVFVPA, NNTDVFVPAV, ANVGRNAFELFLIGSATSDEAKG, NIQAQLTE
QPQVTNGVQGN, TKISDFGSFIGFK, VSVAGGGASQWGN, TLRAGRVANQ
FDDASQAIN, DSNNDVASQLGIFK, GGFSGFSG, LSENGDKAKTKNSTTE
And a recombinant microorganism capable of expressing a peptide selected from the group consisting of VPRISYAHGFDLIERGKKG. 15. A recombinant strain of Neisseria meningitidis capable of expressing a complete meningococcal class I outer membrane protein consisting of any one of the amino acid sequences of claim 1 or a fusion protein thereof. 16. An isolated nucleic acid encoding a full-length Neisseria meningitidis class I outer membrane protein consisting of any one of the amino acid sequences of claim 1. 17. A Neisseria meningitidis class I outer membrane protein 17. The isolated nucleic acid according to claim 16, which comprises the amino acid sequence shown by. 18. The following nucleotide sequence: An isolated nucleic acid consisting of any one of: 19. The following nucleotide sequence: An isolated nucleic acid consisting of any one of: 20. A nucleic acid vector comprising the isolated nucleic acid of claim 19. 21. A nucleic acid encoding a complete meningococcal class I outer membrane protein consisting of any one of the amino acid sequences of claim 1 or a fusion protein thereof, said nucleic acid comprising: Comprises all of the sequences encoding a Bacillus subtilis class I outer membrane protein, wherein the coding sequences are under the control of transcription signals and are linked to appropriate translation signals for expression in a suitable host cell. An expression vector characterized in that 22. A host cell containing the expression vector of claim 21. 23. The method of claim 1, wherein the expression vector of claim 21 is introduced into a suitable host cell and the host cell is cultured under conditions that allow expression of the coding sequence. A method for producing a complete Neisseria meningitidis class I outer membrane protein or fusion protein thereof comprising any one of the described amino acid sequences. 24. A host cell containing a recombinant nucleic acid encoding a Neisseria meningitidis class I outer membrane protein or an immunogenic protein thereof, which comprises any one of the amino acid sequences according to claim 1, and the nucleic acid N. meningitidis class I, characterized in that it is maintained under conditions suitable for the expression of N. meningitidis class I outer membrane proteins encoded thereby or expressed thereby and produced thereby. A method for producing an outer membrane protein or an immunogenic protein thereof. 25. A vaccine comprising a substantially purified native Neisseria meningitidis class I outer membrane protein consisting of any one of the amino acid sequences of claim 1. 26. The vaccine of claim 25, which is free of Neisseria meningitidis class 2 or class 3 outer membrane proteins. 27. The vaccine according to claim 25, further comprising a lipid or a surface-active substance. 28. One or more substantially purified native N. meningitidis class I outer membrane proteins consisting of any one of the amino acid sequences of claim 1 in liposomes or microspheres. A vaccine comprising within.